MD, PhD Nicolas Guibert
email: guibert.n@chu-toulouse.fr

Greg Jones

PhD John F Beeler

PhD Vincent Plagnol

MD d ; Clive Morris

Jean Mourlanette

MD Myriam Delaunay

PhD b ; Laura Keller

MD e ; Isabelle Rouquette

Gilles

Pharm D, PhD b ; Gilles Favre

PhD Anne Pradines

MD, PhD Julien Mazieres

Targeted sequencing of plasma cell-free DNA to predict response to PD1 inhibitors in advanced non-small cell lung cancer

Keywords: next generation sequencing, liquid biopsy, circulating tumor DNA, immunotherapy, PD-1 inhibitors, STK11, PTEN, lung cancer

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

Introduction

The treatment landscape for NSCLC is rapidly changing with the introduction of immune checkpoints inhibitors targeting PD-1/PD-L1 (ICI), but durable clinical benefit to these immune modulating agents is limited to a subset of patients. Furthermore, treatment discontinuation in nonresponders is often delayed because of difficult imaging interpretation. Patient selection is therefore crucial and identifying reliable markers that can quickly predict sustained response is a major challenge. PD-L1 expression, the most validated predictive biomarker, is far from being perfect, due notably to operator interpretation variability [START_REF] Cooper | Intra-and Interobserver Reproducibility Assessment of PD-L1 Biomarker in Non-Small Cell Lung Cancer[END_REF] or its temporal [START_REF] Han | Change in PD-L1 Expression After Acquiring Resistance to Gefitinib in EGFR-Mutant Non-Small-Cell Lung Cancer[END_REF] and spatial [START_REF] Ilie | Comparative study of the PD-L1 status between surgically resected specimens and matched biopsies of NSCLC patients reveal major discordances: a potential issue for anti-PD-L1 therapeutic strategies[END_REF] heterogeneity.

Genomic profiling represents another emerging strong determinant of response to immunotherapy.

Exome analysis of tumors from patients treated with pembrolizumab, demonstrated that the best responses to PD-1 blockade were observed in tumors harboring high tumor mutation burden (TMB), and thus tumor-specific antigen load [START_REF] Rizvi | Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer[END_REF]. Since then, TMB has been shown to be a strong marker of response to front-line treatment with nivolumab in combination with ipilimumab in advanced NSCLC [START_REF] Hellmann | Nivolumab plus Ipilimumab in Lung Cancer with a High Tumor Mutational Burden[END_REF].

However, whole-exome sequencing (WES) or the use of broad NGS panels might be challenging to translate into routine clinical practice due to cost, the lack of a standardized panel and cut-off, the limited availability of tissue and high-quality DNA and required bioinformatics analysis. There is thus a need to move beyond TMB and identify specific genetic determinants of response to PD-1 inhibitors, especially since not all point mutations will result in the genesis of highly immunogenic peptides [START_REF] Chen | Elements of cancer immunity and the cancer-immune set point[END_REF].

Correlations between KRAS mutations and durable response to checkpoint inhibitors have been systematically observed [START_REF] Rizvi | Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer[END_REF][START_REF] Calles | Expression of PD-1 and Its Ligands, PD-L1 and PD-L2, in Smokers and Never Smokers with KRAS-Mutant Lung Cancer[END_REF]. On the contrary, oncogenic drivers found in non-smokers (EGFR, ALK, BRAF V600E) are associated with low tumor burden, resulting in a non-inflamed ("excluded") tumor microenvironment, and low activity of ICI [START_REF] Gainor | EGFR Mutations and ALK Rearrangements Are Associated with Low Response Rates to PD-1 Pathway Blockade in Non-Small Cell Lung Cancer: A Retrospective Analysis[END_REF][START_REF] Mazieres | Efficacy of immunecheckpoint inhibitors (ICI) in non-small cell lung cancer (NSCLC) patients harboring activating molecular alterations (ImmunoTarget)[END_REF]. More interestingly, some molecular alterations have been shown to have a direct impact on the tumor microenvironment. In particular, STK11 mutations, inactivated in ~15% of adenocarcinomas, and often associated with KRAS mutations [START_REF] Schabath | Differential association of STK11 and TP53 with KRAS mutation-associated gene expression, proliferation and immune surveillance in lung adenocarcinoma[END_REF], have been shown to be associated with non-inflamed tumor microenvironment and reduced PD-L1 expression [START_REF] Koyama | STK11/LKB1 Deficiency Promotes Neutrophil Recruitment and Proinflammatory Cytokine Production to Suppress T-cell Activity in the Lung Tumor Microenvironment[END_REF]. In contrast, tumors mutated for both KRAS and TP53 demonstrate increased PD-L1 expression along with prominently increased mutation burden that is specifically enriched in a transversion-high phenotype and show stronger inflammatory responses than tumors harboring KRAS and STK11 mutations [START_REF] Schabath | Differential association of STK11 and TP53 with KRAS mutation-associated gene expression, proliferation and immune surveillance in lung adenocarcinoma[END_REF][START_REF] Dong | Potential Predictive Value of TP53 and KRAS Mutation Status for Response to PD-1 Blockade Immunotherapy in Lung Adenocarcinoma[END_REF][START_REF] Skoulidis | STK11/LKB1 Mutations and PD-1 Inhibitor Resistance in KRAS-Mutant Lung Adenocarcinoma[END_REF]. These preclinical results have considerable clinical relevance, with KRAS/STK11 tumors being associated with primary resistance to PD-1 inhibitors (0 to 7.4% ORR), compared to KRAS/TP53 tumors (35.7% to 57% ORR) [START_REF] Skoulidis | STK11/LKB1 Mutations and PD-1 Inhibitor Resistance in KRAS-Mutant Lung Adenocarcinoma[END_REF]. Similarly, loss of PTEN results in decreased T-cell infiltration and increased immunosuppressive cytokines expression and poor outcomes under ICI [START_REF] Peng | Loss of PTEN Promotes Resistance to T Cell-Mediated Immunotherapy[END_REF]. These correlations of highly prevalent genetic events with response to ICI are of great interest and pave the way towards the use of more targeted sequencing assays.

The lack of tumor tissue, the heterogeneous pattern of PD-L1 expression and of the dynamic molecular landscape through treatment (with a few acquired mutations (JAK2, B2M) identified as mechanisms of resistance to these agents [START_REF] Zaretsky | Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma[END_REF][START_REF] Shin | Primary Resistance to PD-1 Blockade Mediated by JAK1/2 Mutations[END_REF]) render non-invasive approaches valuable to predict and monitor response to ICI. While liquid biopsy applications has been widely studied in the targeted therapy space, its potential to guide and follow response to immune therapy is just beginning to be investigated [START_REF] Cabel | Clinical potential of circulating tumour DNA in patients receiving anticancer immunotherapy[END_REF]. Several reports suggest potential of cell-free DNA in assessing TMB [START_REF] Gandara | Blood-based tumor mutational burden as a predictor of clinical benefit in non-small-cell lung cancer patients treated with atezolizumab[END_REF] and monitoring response [START_REF] Guibert | Monitoring of KRAS-mutated ctDNA to discriminate pseudo-progression from true progression during anti-PD-1 treatment of lung adenocarcinoma[END_REF][START_REF] Cabel | Circulating tumor DNA changes for early monitoring of anti-PD1 immunotherapy: a proof-of-concept study[END_REF][START_REF] Goldberg | Early Assessment of Lung Cancer Immunotherapy Response via Circulating Tumor DNA[END_REF]. This last point is particularly appealing because TMB can only be assessed in approximately 60% of tissue specimens (5), providing clinical utility for blood-based approaches covering situations where no adequate tissue is available. However, large gene panels are required for bTMB and if low tumor cell content in tissue samples is a known confounding factor in assessment of TMB [START_REF]PD Analytic validation of tumor mutational burden as a companion diagnostic for combination immunotherapy in non-small cell lung cancer[END_REF], with cell-free DNA, the ctDNA fraction is dependent on tumor shedding and on the amount of normal DNA in the plasma and thus is especially variable. Low levels of ctDNA may not allow for an accurate determination of the TMB status of a tumor.

Focusing on specific molecular events known to be associated with response to ICI, reported above, we investigated the utility of a plasma genotyping to stratify patients between durable responders and non-responders, as well explore if early changes in ctDNA AF can serve as a valid pharmacodynamics biomarker to predict sustained response.

Methods

Patients

Patients with stage IIIB/IV, progressive NSCLC, consented to receive PD1 inhibitors were included in the study. Among 187 patients from the IMMUNOPREDICT trial (NCT02827344, blood collection including plasma, circulating tumor cells and peripheral blood mononuclear cells from patients treated by ICI in second line setting), "responders" (Objective response on first evaluation and PFS > 6 months) and "non-responders" (progressive disease at first CT evaluation) patients were selected. Plasma specimens were collected after informed consent, prior to initiation of ICI and when feasible, at 1 month. Plasma analyses were performed blinded to clinical information such as tumor histology, genotype, smoking history, PD-L1 status, or response to ICI.

Plasma NGS

Amplicon-based plasma NGS was performed by Inivata (Morrisville, NC), using the InVision First ® -Lung technology [START_REF] Guibert | Amplicon-based nextgeneration sequencing of plasma cell-free DNA for detection of driver and resistance mutations in advanced non-small cell lung cancer[END_REF]. 36 cancer-related genes were sequenced using gene specific primers designed to hotspots and entire coding regions of interest (Supp. Figure 1). NGS libraries are prepared from 2,000 -16,000 amplifiable copies of the genome using a two-step PCR amplification process incorporating replicate and patient-specific barcodes and Illumina sequencing adaptors. Samples are quantified and pooled to generate a normalized library of 12 nM. 1.8 pM libraries are sequenced on the Illumina NextSeq 500. Sequencing files were analysed using Inivata's proprietary Somatic Mutation Analysis (ISoMA) and FUSP pipelines.

Monitoring of response using ctDNA kinetics

An early blood draw (before the second cycle of nivolumab, median 28 days) was studied in 65 patients to monitor the early kinetics of plasma ctDNA burden through treatment. At baseline, a single somatic change (a tumor driver when present, or the variant with the highest allelic frequency) was chosen for subsequent monitoring.

Tissue PD-L1 expression

PD-L1 expression on tissue was assessed by immunohistochemistry using an anti-PD-L1 rabbit monoclonal antibody (clone E1L3N, Cell signalling Technology).

Statistical analysis

Statistical analysis was performed with MedCalc Statistical Software ver 18.10. Specific statistical tests are indicated. Sensitivity of plasma NGS for detection of driver and resistance mutations were calculated using clinically performed tumor genotyping as reference standard. The median survival time and its 95% CI is calculated according to Brookmeyer et al [START_REF] Brookmeyer | A Confidence Interval for the Median Survival Time[END_REF]. Results of the logrank test are used in the comparison of survival curves.

Results

Detection of known driver and resistance mutations in ctDNA

Samples from 42 responders and 55 non-responders patients were available and analyzed.

Characteristics of the patient cohort are listed in Table 1. 86 specimens were evaluable for a baseline molecular profile (7 NR patients received less than 2 cycles due to toxicity and were excluded from analysis and 4 runs failed). Of these 86 baseline samples, at least one variant was detected in 67/86 samples (78% sensitivity). Using tissue genotyping as a reference standard, sensitivity was 74% (25/34) for the detection of KRAS mutations and 100% for EGFR mutations (6/6, 2/2 exon 19 deletion, 1/1 T790M, other 3/3) and MET amplifications (2/2). The only ALK fusion specimen tested using the fusion assay was not detected. Two ALK resistance mutations (G1202R and F1174C) were detected in another ALK patient with acquired resistance to crizotinib, in agreement with tissue.

Targeted profiling of cfDNA to predict outcomes under PD-1 inhibitors: Correlation of an algorithm for prediction of response

Of the 86 evaluable patients, 39 patients exhibited clinical benefit to ICI (PFS>6 months) while 47 patients exhibited progressive disease. Based on preclinical and clinical observations reported in the introduction, we tested the ability of specific molecular events to stratify responders and nonresponders. Median PFS for the overall evaluable population (n=86) was 7 months. Patients with no detectable ctDNA had a 3.5 months PFS (progression-free survival) and were considered nonevaluable with regards to the predictive algorithm. In agreement with previous reports [START_REF] Gainor | EGFR Mutations and ALK Rearrangements Are Associated with Low Response Rates to PD-1 Pathway Blockade in Non-Small Cell Lung Cancer: A Retrospective Analysis[END_REF][START_REF] Mazieres | Efficacy of immunecheckpoint inhibitors (ICI) in non-small cell lung cancer (NSCLC) patients harboring activating molecular alterations (ImmunoTarget)[END_REF], patients with a targetable alteration detected in blood (1/1 ALK, 5/5 EGFR) progressed on the first clinical evaluation (PFS 2 months). All of them had received multiple targeted therapies before ICI.

Patients with a PTEN or a STK11 mutation derived poor outcomes compared with patients without (HR 8.9, p=0.09 for PTEN; HR 4.7, p=0.003 for STK11; Figure 1A). In contrast, patients with a KRAS Tv mutation or a TP53 Tv mutation derived better outcomes compared to patients without (HR 0.36, p=0.011 for TP53 Tv; HR 0.46, p=0.11 for KRAS Tv; Figure 1B). Median PFS for each cohort are detailed in Figure 1.

An algorithm was built blinded to patients' results combining these molecular alterations known to be associated with good or poor outcomes. A "High immune score" was defined as follow: no targetable driver (EGFR, ROS1, ALK, BRAF V600E), no PTEN or STK11 mutations, but a transversion mutation in KRAS or TP53. A "Low Immune Score" was defined as follow: presence of a targetable alteration, a PTEN or STK11 mutation or in the absence of KRAS or TP53 transversion mutation) (Figure 1C). Patients with "High Immune Score" had a median PFS of 14 months (HR 2.89, 95%CI 1.63-5.83) compared to 2 months (95%CI 1.5-months, p=0.0008) in the "Low Immune Score" (Figure 1D). 6-month PFS was 76% vs 33% for those with high immune score vs low immune score (p=0.0006).

Comparison with tissue PD-L1 expression

PD-L1 expression was not statistically associated with response to ICI, using either a cutoff of 1% (HR 1.73, 95%CI 0.93-3.23) or 50% (HR 1.35, 95%CI 0.73-2.49), and was less predictive of response than ctDNA profiling (Sup. Figure 3).

Early detection of response through serial plasma NGS

Patients with any decrease of ctDNA AF at one month had longer PFS (median 10 (95%CI 8-18) months) than patients with early increase (median PFS 2 months (95%CI 1.5-ND)). 74% of patients with early molecular response were still responding to treatment after 6 months compared to 16% of patients with plasma increase (Figure 2A). Using cut-offs of 50% or 30% for AF variations to define molecular response or progression, correlation between early ctDNA kinetics and clinical response was stronger, with only 11% and 6.3% of patients with plasma progressions still responding at 6 months for 30 and 50% thresholds, respectively (Figure 2B and2C).

Discussion

TMB, measured whether in tissue or blood, is correlated with response to ICI but integration into routine clinical practice is challenging due to lack of tissue availability, high costs associated with WES or broad panels exhibit high and time-consuming approaches. Furthermore, less than 10% of non-synonymous mutations will harbor MHC class I bindings motifs capable of recognition by T cells as neoantigens [START_REF] Chen | Elements of cancer immunity and the cancer-immune set point[END_REF][START_REF] Miao | Genomic correlates of response to immune checkpoint blockade in microsatellite-stable solid tumors[END_REF].

In parallel of efforts to develop TMB analysis platforms, an increasing number of specific alterations that correlates to ICI outcomes have been investigated, suggesting an opportunity to use more restrictive sequencing assays. A good concordance between targeted NGS covering more than 200 genes and WES for TMB evaluation was demonstrated [START_REF] Rizvi | Molecular Determinants of Response to Anti-Programmed Cell Death (PD)-1 and Anti-Programmed Death-Ligand 1 (PD-L1) Blockade in Patients With Non-Small-Cell Lung Cancer Profiled With Targeted Next-Generation Sequencing[END_REF], demonstrating the ability of NGS panels to stratify patients between responders and non-responders with high accuracy. We herein show that more limited, carefully selected genes panel can accurately predict response to ICI, using a simple algorithm. Moreover, this molecular profiling was done in plasma, offering a rapid, tissue-sparing and repeatable access to the tumor genomics. If previous studies have demonstrated that TMB can be accurately estimated in cfDNA (blood TMB, bTMB) with good concordance with tissue and response to ICI [START_REF] Gandara | Blood-based tumor mutational burden as a predictor of clinical benefit in non-small-cell lung cancer patients treated with atezolizumab[END_REF]; a more targeted analysis of cfDNA focusing on specific alterations known to alter or increase response to ICI has never been investigated.

Interestingly, patients with no detectable ctDNA tended to experience poor outcomes (PFS 3.5 months), even though the presence of ctDNA is usually of detrimental prognostic impact. The limited number of patients does not allow for definitive conclusion, but the absence of ctDNA could be an indirect indication of low TMB. This is consistent with what was observed using bTMB, the "biomarker-evaluable population" (patients with MAF >1%) experiencing the best outcomes [START_REF] Gandara | Blood-based tumor mutational burden as a predictor of clinical benefit in non-small-cell lung cancer patients treated with atezolizumab[END_REF]. We here confirm the significant impact of the subtype of mutations (transversion vs transitions) on ICI outcomes, a phenomenon already clearly demonstrated in tissue [START_REF] Rizvi | Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer[END_REF][START_REF] Miao | Genomic correlates of response to immune checkpoint blockade in microsatellite-stable solid tumors[END_REF] but not used routinely in clinical practice nor on a specific group of genes. This work also shows that the detrimental effect of STK11 and PTEN mutations on ICI outcomes reported in tissue is relevant in liquid biopsy. The combination of all these specific markers, along with known targetable oncogenic drivers, allows guiding patient treatment towards either targeted therapy or ICI (depending on the molecular event(s) detected) with one and only, non-invasive and rapid assay.

We also show that early kinetics of ctDNA burden strongly predicts durable responses, as previously demonstrated in 2 reports where a complete [START_REF] Goldberg | Early Assessment of Lung Cancer Immunotherapy Response via Circulating Tumor DNA[END_REF] or partial [START_REF] Cabel | Circulating tumor DNA changes for early monitoring of anti-PD1 immunotherapy: a proof-of-concept study[END_REF] ctDNA clearance at 8 weeks was strongly predictive of sustained response. These observations are of great value in the space of immune therapy, early radiological changes being challenging to interpret. Plasma genotyping has the potential to early distinguish pseudo from true progression during ICI treatment, as reported in limited series [START_REF] Guibert | Monitoring of KRAS-mutated ctDNA to discriminate pseudo-progression from true progression during anti-PD-1 treatment of lung adenocarcinoma[END_REF][START_REF] Lee | Association Between Circulating Tumor DNA and Pseudoprogression in Patients With Metastatic Melanoma Treated With Anti-Programmed Cell Death 1 Antibodies[END_REF]. Other applications of cfDNA are likely to emerge, such as the detection of MRD for adjuvant immunotherapy, or detection of mechanisms of resistance, like acquired JAK1/2 or B2M mutations [START_REF] Cabel | Clinical potential of circulating tumour DNA in patients receiving anticancer immunotherapy[END_REF].

The first major limitation of this study is its retrospective nature. Even though the ctDNA analysis was performed blinded to clinical results, the study was enriched in responders' patients (45% of the population), partially explaining the unusual PFS in the overall population (7 months) and in the "High immune score" cohort (14 months). These promising data will need to be validated prospectively in larger and unselected populations. A second limitation is the lack of a non-ICI treatment arm to discriminate a true predictive value from a prognostic effect of the algorithm. However, KRAS [START_REF] Villaruz | The prognostic and predictive value of KRAS oncogene substitutions in lung adenocarcinoma[END_REF] and TP53 (29) are known to be of detrimental effect in NSCLC, whereas oncogenic drivers like EGFR mutations [START_REF] Izar | The impact of EGFR mutation status on outcomes in patients with resected stage I non-small cell lung cancers[END_REF] or ALK fusion (31) are of better prognosis. The opposite results observed in this work are thus very likely due to a predictive value of these alterations more than to a simple prognostic impact.

Another limitation of our study is the incomplete tissue genotyping data available. Although we could test and demonstrate a good concordance with tissue for major driver alterations, no tissue was left to analyze other alterations such as PTEN, STK11 or TP53. However, this is, in our opinion, an interesting point of our study, demonstrating that ctDNA can be a powerful tool to identify biomarkers when no tissue is available (a frequent situation in NSCLC where there is a current paradox between the development of minimally invasive diagnostic tools leading to small samples and the multiplication of markers for targeted therapy and ICI. The concordance of the plasma genotyping platform used in this work with tissue has however been studied in a prospective manner in another study and appeared very high (97.8%; with 82.9% positive predictive value, 98.5% negative predictive value, 70.6% sensitivity, and 99.2% specificity) [START_REF] Pritchett | Prospective Clinical Validation of the InVisionFirst-Lung Circulating Tumor DNA Assay for Molecular Profiling of Patients With Advanced Nonsquamous Non-Small-Cell Lung Cancer[END_REF].

In conclusion, plasma NGS using limited panel can accurately predict sustained responses under PD-1 blockade. This approach has the unique ability to select patients for either genotype-directed therapies or ICI using one unique assay, in particular when tissue is not available. Algorithm genesis: If a targetable oncogenic driver is detected, the probability of response is low and targeted therapies (or chemotherapy if already received) must be favored over immunotherapy. The presence of PTEN or STK11 mutations also predicts primary resistance to ICI, while transversion mutations in KRAS, TP53 or other genes ("immune high" somatic signature) is associated with good outcomes. 1D: Correlation of the algorithm with outcomes: An "Immune High Score", defined by the absence of targetable driver, PTEN or STK11 mutation but the presence of KRAS and/or TP53 mutations, strongly predict favorable outcomes under PD-1 inhibitors (p=0.0008).

Figure and Table legends: Table 1: Characteristics of the population

Figure 2:

Early changes in ctDNA level to predict sustained responses under PD-1 inhibitors. 2A: A decrease in ctDNA burden at one month is strongly predictive of durable response (74% PFS at 6 months), while an early increase indicates progression (84% progressive disease at 6 months). Using cut-offs of 30% (2B) and 50% (2C) for allelic fractions changes enhances this predictive effect.

Figure 1 :

 1 Figure 1: Proposed algorithm of interpretation of cfDNA molecular profile in order to guide ICI initiation. 1A: Patients harboring STK11 or PTEN mutations derive poor benefit from PD-1 inhibitors compared with patients who don't (HR 4.7, p=0.003 for STK11; HR 8.9, p=0.09 for PTEN). 1B: Patients with a KRAS or TP53 transversion mutation showed better responses compared to patients without (HR 0.36, p=0.011 for TP53 Tv; HR 0.46, p=0.11 for KRAS Tv). 1C:

  Median PFS TP53 (27)= 10 months (95%CI 7-14) KRAS (10) = 7.5 months (95%CI 2-24) Neither (23) = 2 months (95%CI 1.5-ND) Immune High (n=31) = 14 months (95%CI 9 -24) Immune Low (n=35) = 2 months (95%CI 1.5 -ND) Undetectable (n=20) = 3.5 months (95%CI 2 -4)

17

 17

( 1 .

 1 775 to 9.811) p<0.0001 2B Median PFS Decrease > 30% (n=23) = 14 months (95%CI 9-14) Increase > 30% (n=19) = 2 months (95%CI 1.5-ND)

informed consent (IMMUNOPREDICT trial (NCT02827344This research was supported in part by Inivata, "La Ligue contre le Cancer" and Toulouse University Hospital (DRCI: Direction de la Recherche Clinique et de l'Innovation). Author's contributions: NG: Principal Investigator of the work: Conception and design of the work. Acquisition and interpretation of data. Drafting manuscript. GJ, VP, CM and the work. Acquisition and interpretation of data.

of data and materials: The datasets during and/or analyzed during the current study are available from the corresponding author on reasonable request. Plasma specimens and clinical information's were collected after


Isabelle Fabre-Francke

Mickaël Berrebi

Bertrand Lavédrine

Odile Fichet
email: odile.fichet@u-cergy.fr

Hybrid PMMA combined with Polycarbonate inside interpenetrating polymer network architecture for development of new anti-scratch glass

Keywords: Polycarbonate, Polymethyl methacrylate, Hybrid interpenetrating polymer networks (hybrid IPNs), anti-scratch properties

Polymethyl methacrylate (PMMA) is commonly used as organic glass for the protection of works in museums. However, their extreme sensitivity to scratching causes problems during maintenance procedures. In order to improve the resistance to abrasion, PMMA has been cross-linked with silica nanoparticles previously modified with 3-(methacryloxy)propyl-trimethoxysilane (MPTS). This functionalization allows both a homogeneous dispersion of the silica nanoparticles in MMA monomer and the PMMA crosslinking which has been confirmed by solid/liquid extraction and DMA analysis. The small nanoparticles (7 -10 nm diameter) added in different amounts, are homogeneously dispersed in the transparent (T% > 80%) hybrid PMMAx-SiO2 networks as verified by TGA and TEM microscopy. These hybrid PMMAx-SiO2

Introduction

Polymethyl methacrylate (PMMA) is used in numerous applications because of its optical properties close to those of glass. Indeed, the refractive indices of PMMA and glass are of 1.491 [START_REF] Brandrup | Polymer Handbook[END_REF] and 1.522 [START_REF] Snitzer | Optical maser action of Nd 3+ in a barium crown glass[END_REF], respectively. PMMA also exhibits a better light transmission, is significantly (1.19 g.cm -3 by respect to 2.5 g.cm -3 ) and less brittle than glass. However, PMMA exhibits poor resistance to scratching [START_REF] Bj | An experimental study of the nanoscratch behaviour of poly (methylmethacrylate)[END_REF][START_REF] Pelletier | Viscoelastic and elastic-plastic behaviors of amorphous polymeric surfaces during scratch[END_REF] .

To enhance the PMMA's scratch resistance, it was combined with polycarbonate (PCR39 ® ) in interpenetrating polymer network (IPN) architecture. An IPN is defined as a combination of at least two cross-linked polymers, one of being synthesized in the presence of the other [START_REF] Lh | Interpenetrating polymer networks[END_REF]. Thus, a series of transparent IPNs (transmission higher than 95%) based on the combination of an aliphatic polycarbonate PCR39 ® and PMMA was synthesized without solvent according to in situ polymerizations [START_REF] Berrebi | Development of organic glass using Interpenetrating Polymer Networks with enhanced resistance towards scratches and solvents[END_REF]. The scratch tests revealed that the addition of only 25 wt% PCR39 ® in PMMA network is sufficient to improve the PMMA's scratch resistance.

An alternative route to improve scratch resistance of polymer material is based on the incorporation of inorganic compounds. For instance, Bauer et al. [START_REF] Bauer | Preparation of scratch and abrasion resistant polymeric nanocomposites by monomer grafting onto nanoparticles, 3 a Effect of filler particles and grafting agents[END_REF] have studied the effect of nanoparticles functionalized by a trialkoxysilane with a terminal vinyl function on the anti-abrasive properties of a polyacrylate-based UV topcoat for parquet (SR-494/PHEMA). Authors have shown that abrasion is significantly more decreased with the introduction of functionalized alumina, silica or zirconium than that of modified titanium nanoparticles. The key role of the functionalization of nanoparticles [START_REF] Hansen | Hansen Solubility Parameters: A user's Handbook[END_REF][START_REF] Hildebrand | The Solubility of Nonelectrolytes[END_REF][START_REF] Fukao | Dynamics of α and β processes in thin polymer films: Poly(vinyl acetate) and poly(methyl methacrylate)[END_REF](33)(34)(35)(36)(37)(38)(39)(40) nm diameter) on the abrasion resistance has also been confirmed by the same authors on the coating of SR 494. Indeed, the SR 494 topcoat containing 35 wt% nanoparticles functionalized with 3-(methacryloxy)propyl-trimethoxysilane (MPTS) is 4 times more scratch resistant than the coating alone in which less than 15 wt% unmodified silica can be included [START_REF] Gläsel | Preparation of scratch and abrasion resistant polymeric nanocomposites by monomer grafting onto nanoparticles, 2-characterization of radiation-cured polymeric nanocomposites[END_REF][START_REF] Bauer | UV Curable Acrylate Nanocomposites: Properties and Applications[END_REF].

The aim of our research project is focused on the development of a hybrid interpenetrating polymer network based on organic PMMA/PCR39 ® IPN. The first challenge is generally the control of the homogeneous dispersion of the filler within the polymer matrix. Thus, silica in situ synthesized from tetraethoxysilane or trimethoxysilane is used almost exclusively as inorganic phase [START_REF] Mw | Mutually interpenetrating inorganic-organic networks. New routes into nonshrinking sol-gel composite materials[END_REF][START_REF] Aej | Transparent silica gel-PMMA composites[END_REF]. In this work, exogenous silica nanoparticles have been selected because of their capability to be dispersed in both PMMA-based and polycarbonate-based materials.

The main objectives of the nanoparticle addition in PMMA generally is to increase its glass transition temperature (Tg), its mechanical modulus and/or its thermal resistance. For instance, PMMA's Tg is increased from 106°C to 122°C when TiO2 nanofibers modified with methacrylic acid are incorporated from 0 wt% to 17 wt% [START_REF] Sm | Synthesis of TiO2-PMMA Nanocomposite: Using Methacrylic Acid as a Coupling Agent[END_REF]. However, when the fillers are not functionalized, the Young's modulus is decreased and Tg does not evolve steady. This suggests the importance of the functionalization of the inorganic fillers for their integration within PMMA materials. Furthermore, Otsuka et al. [START_REF] Otsuka | Poly(methyl methacrylate) (PMMA)-based hybrid materials with reactive zirconium oxide nanocrystals[END_REF] have functionalized 5 nm zirconium oxide nanoparticles with MPTS in order to disperse them in methylmethacrylate (MMA) monomer. The authors show that the incorporation of up to 39 wt% ZnO2 functionalized nanoparticles leads to the preparation of transparent PMMA networks with improved thermal resistance (degradation temperature -Td=280°C) compared to uncharged PMMA (Td=200°C).

Silica is also often used as nanoparticles. They were previously modified with allyl glycidyl ether [START_REF] Liu | Poly(methylmethacrylate)-silica nanocomposites films from surface-functionalized silica nanoparticles[END_REF] or with 3-methacryloxypropyl dimethylchlorosilane [START_REF] Ps | Synthesis of poly(methyl methacrylate)-silica nanocomposites using methacrylate-functionalized silica nanoparticles and RAFT polymerization[END_REF], for example. Thus, the synthesis of hybrid PMMA network was carried out with from 10 to 23 wt% of 20 nm silica nanoparticles modified with 3-methacryloxypropyl dimethylchlorosilane by quasi-living polymerization. No nanoparticle aggregation was observed and the PMMA mechanical relaxation temperature Tα was risen by approximately 10°C.

Simultaneously a slight increase in storage and loss moduli of the rubber plateau was detected. These results therefore indicate that PMMA was crosslinked by the silica nanoparticles under these conditions. While hybrid material based on PMMA are widely described, few of these studies care about PMMA scratch resistance. On the one hand, Avella et al. [START_REF] Avella | Novel PMMA/CaCO3 nanocomposites abrasion resistant prepared by an in situ polymerization process[END_REF] have shown that the PMMA's anti-abrasive properties are improved (a weight loss two times less was measured for a similar abrasion) when 6wt% CaCO3 nanoparticles (about 40 nm diameter functionalized with stearic acid) are added. On the other hand, Rodriguez et al. [START_REF] Rodriguez | Hybrid ceramic-polymer material for wood coating with high wearing resistance[END_REF] have inserted up to 30 wt% silica nanoparticles functionalized with dichlorodimethylsilane in PMMA. They show that 50 µm coating spread on wood reduces by four the weight loss during abrasion compared to that of the commercial coating (name and composition not specified). However, in this case, nanoparticles are not grafted on PMMA and can thus migrate in organic matrix over time.

Polycarbonate (PC) has also been studied as organic matrix of hybrid materials. For instance, it has been associated with zinc oxide nanoparticles to improve its wear resistance [START_REF] Fj | Influence of ZnO nanoparticle filler on the properties and wear resistance of polycarbonate[END_REF]. The introduction of only 0.5 wt% of non-functionalized nanoparticles increases its hardness and thus the hybrid material wear during an abrasion test is lower than that of pure PC. However, the material heterogeneity simultaneously leads to the decrease of thermal stability. In addition, the incorporation of ZnO nanoparticles in polycarbonate matrix increases the material brittleness at room temperature by a factor of 2.72 [START_REF] Md | Scratch resistance of polycarbonate containing ZnO nanoparticles: effects of sliding direction[END_REF]. Other nanoparticles such as CaCO3 or aluminum nanofibers were introduced in polycarbonate to improve its mechanical properties. However, a transparency loss was observed beyond 0.5 wt% fillers (transmission at 450 nm less than 80%) due to the charge's agglomeration [START_REF] Wang | Rheology enhancement of polycarbonate/calcium carbonate nanocomposites prepared by meltcompounding[END_REF][START_REF] Hr | Synthesis and characterization of transparent alumina reinforced polycarbonate nanocomposite[END_REF]. All these results highlight either a loss of transparency or a degradation of the mechanical or thermal properties of filled PC. The effect of the introduction of silica nanoparticles on the polycarbonate scratch resistance was also studied by coating but not in bulk. For that, a mixture of polycarbonate with 20 wt% silica was deposited on the PC surface and covalent bonds are created between the polymer and the fillers by a sol-gel process method [START_REF] Zz | Indentation and scratch behavior of nano-SiO2/polycarbonate composite coating at the micro/nano-scale[END_REF]. The depth of scratches was less important when the PC is previously modified with a PC-SiO2 coating whatever its thickness rather than a single PC film. In addition, this PC-SiO2 coating induces an increase in the hardness, the elastic modulus as well as the scratch recovery capability.

Hybrid organic-inorganic UV-cured coatings based on interpenetrating polymer networks were finally synthetized from bisphenol-A-diglycidyl ether dimethacrylate / diglycidyl ether of bisphenol A in the presence of 15wt% fumed silica nanoparticles [START_REF] Lungu | Thermo-mechanical properties of interpenetrating polymer networks (IPNS) filled with fumed silica nanoparticles[END_REF]. An improvement of 34% of compression strenght was obtained for (50/50) hybrid IPNs.

As the nanoparticle dispersion seems easier in PMMA than in polycarbonate, we focused our work on the development of a series of hybrid PMMA/PCR39 ® IPNs in which PMMA would be cross-linked by silica nanoparticles. For that, the surface of the silica nanoparticles has been first functionalized with methacrylate functions to help their copolymerization with MMA monomers. Hybrid PMMA networks were then first synthesized and characterized. These hybrid networks were included in IPN architecture with PCR39 ® polycarbonate to obtain homogeneous transparent materials with a scratch resistance higher than those of corresponding organic materials were.

Experimental Section

Materials

Diethylene glycol bis-allyl carbonate (CR39 ® ) (Aldrich), methyl methacrylate (MMA) (Acros organics 99%), dicyclohexyl peroxidicarbonate (DCPD) (group Arnaud), 3-(methacryloxy)propyl-trimethoxysilane (MPTS) (ABCR), methanol (VWR) and dichloromethane (GPR Rectapur ® VWR) were used as received. SiO2 nanoparticles of 4.1±1.3 nm diameter were furnished (ABCR) in pH 10.0 aqueous solution at 15%wt SiO2 concentration. Benzoyl peroxide (BPO) (Aldrich 75%) was dried under vacuum at room temperature for 24h before use.

Preparation of MPTS-modified SiO2 as cross-linker

The functionalization of SiO2 nanoparticles was carried out with 6.67g of SiO2 aqueous solution (1g SiO2) in which from 0.12 to 0.35g MPTS (according to the expected grafting ratio) and 20 mL methanol were added. The molar ratio between MPTS and silica nanoparticles was thus between 50 and 150. The transparent methanol/water solution was then shaken under 130 W ultrasonic bath for 10 min and heated at reflux with vigorous stirring for 15h. Methanol was then removed with a vacuum rotary evaporator and a thick and white solution was obtained. For the nanoparticle characterizations, this solution was dried at 90°C for 1h to remove residual methanol and MPST (less than 2% determined by TGA).

For the material synthesis, the previous solution was extracted with 0.8 equivalent weight of MMA in which MPTS-modified SiO2 nanoparticles spontaneously migrate. Thus, a MMA solution containing 1% nanoparticles (determined by TGA measurements) was obtained. To increase the proportion of functionalized nanoparticles, the solution was concentrated with a rotary evaporator from 1 to 13 wt% (also determined by TGA measurement). When the proportion becomes higher than 13wt%, the MMA solution clouded.

Synthesis of hybrid PMMAx-SiO2 network

Single PMMA networks were synthesized starting from the previous MMA solutions containing different proportions of MPTS-modified SiO2. They were prepared by mixing 1g MPTS-modified SiO2/MMA solution and 5mg (30 µmol) DCPD free radical initiator (0.5 wt% with respect to MMA). The mixture was degassed under argon and then poured into the mould made from two glass plates clamped together and sealed with 1mm thick Teflon ® gasket. The mould was then kept at 35°C for 3h and post-cured at 80°C for 1h. Rigid transparent materials were obtained. They are noted PMMAx in the text where x is the nanoparticle weight proportion in the precursor MMA solution. All synthesized materials are transparent.

Synthesis of hybrid PMMAx-SiO2 / PCR39 ® IPNs

Hybrid PMMA13-SiO2 / PCR39 ® (50/50) IPN was synthesized as follows: 0.5g of MMA solution containing 13 wt% MPTS-modified SiO2 was mixed with 0.5g (1.8mmol) CR39 ® . Then 2.5mg DCPD (15µmol -0.5 wt% with respect to MMA) and 25mg BPO (0.1mmol -5 wt% with respect to CR39 ® ) were added. The solution was degassed under argon and then poured into the mould previously described. The mould was heated in an oven at 35°C for 3h, at 80°C for 2h and 100°C for 1h. After mould removal, hybrid PMMA13-SiO2 / PCR39 ® (50/50) IPN is transparent.

IPNs with different PCR39 ® network contents ranging from 25 to 75wt% were synthesized keeping the same proportions between BPO initiator and CR39 ® (5 wt% BPO) and between DCPD initiator and MMA (0.5 wt%). Whatever their composition, all synthesized IPNs are transparent.

Synthesis of semi PMMAx / PCR39 ® IPNs

Semi PMMA / PCR39 ® IPNs were synthesized under the same conditions as corresponding hybrid PMMAx-SiO2 / PCR39 ® IPNs except MMA solution with nanoparticles was replaced by pure MMA.

Polymerization and polycondensation of MPTS

MPTS radical polymerization was carried out by adding 5mg DCPD (0.5 wt% with respect to the MPTS) to 1g MPTS. The mixture was degassed 15 min under argon and then inserted into a test tube. The thermal program was applied from 3h to 35°C and then from 1h to 80°C.

PolyMPTS was formed by hydrolysis reaction followed by condensation of the methoxy functions of MPTS. For that, 1g MPTS in 20 mL methanol were placed in a flask and heated at reflux with vigorous stirring for 15h. Then, methanol was removed with a rotary evaporator.

Characterizations

Nanoparticle size distribution was determined in colloid solution with Zetasizer NanoS PCS (Photon Correlation Spectroscopy) from Malvern Instrument at 25°C.

In order to check the network formations, single networks, semi-IPNs and IPNs were extracted in a Soxhlet with dichloromethane for 72 h. After extraction, the sample was dried under vacuum and then weighted. The extracted content (EC) was given as a weight percentage (Eq.1.):

EC= w 0 -w E w 0 × 100 Equation 1.
where WO and WE are the weights of samples before and after extraction, respectively.

Each extraction was performed three times and the average value was given. The soluble fractions were then analyzed by 1 H NMR using the Brüker Avance DPX250.

The molecular weight between crosslinks (Mc) was measured by swelling the PMMAx-SiO2 networks with dichloromethane. The networks were immersed up to have a constant mass. The uptake was calculated as (Eq.2.):

Uptake % = w after -w before w before × 100 Equation 2.

where wbefore and wafter are the sample weights before and after immersion.

Thermogravimetric analyses (TGA) were performed on a Q50 model (TA Instruments) under synthetic air (60mL.min -1 ) from room temperature to 700°C at 20°C.min -1 heating rate.

Dynamic thermomechanical analysis (DMA) measurements were carried out with a Q800 apparatus (TA instruments) operating in tension mode. Experiments were performed at a frequency of 1Hz and heating rate of 25°C.min -1 from 25 to 200°C.

Typical dimensions of the samples were 8 mm X 20 mm X 1 mm. The damping parameter or loss factor is defined as tan δ = E"/E'.

UV-visible spectra were recorded in absorbance mode with a JASCO V-570 spectrophotometer. The scan rate was set at 400 nm.min -1 between 250 and 800 nm.

The thickness of the samples was about 250 µm or 1 mm.

Fourier transform infrared (FTIR) spectra were obtained using a Brüker spectrometer (Equinox 55) in the range 4000-500 cm -1 by averaging of 16 consecutive scans with a 2 cm -1 resolution.

Transmission electron microscopy (TEM) was performed with a Hitachi H-800 at 200

KV with a resolution of 0.2 nm on ultrafine cuts (about 40 nm thickness) of the samples.

The contrast between the organic and inorganic phases was enhanced by the inorganic phase stained with osmium tetroxide.

The scratch tests were performed on a Scratch Hardness Tester Erichsen model 291.

The pencil (8H hardness-grade -ISO 15184) was moved scratching over the surface under a 45° angle with a 7.5N pressure. The scratch depth was measured with a Veeco Dektak 150 profilometer.

Artificial UV ageing

The artificial UV ageing of the samples was carried out in a Xenotest 220/220 + ageing chamber (ATLAS) equipped with a Xenon lamp equipped with a Xenochrome 320 filter to reproduce the solar spectrum behind a 3-mm thick glass window. The samples were placed in this climatic chamber at 50% RH, 25°C and irradiated at 50 W.m -2 up to 548 hours. The samples were characterized before and after aging by UV-visible spectrometry.

Results and discussion

Characterizations of MPTS-modified SiO2 nanoparticles:

First, the hydroxyl density on the surface of SiO2 nanoparticles has been quantified by thermogravimetric analysis from loss weight starting from 120°C due to their deterioration [START_REF] Mueller | H surface density of SiO2 and TiO2 by thermogravimetric analysis[END_REF]. A value of 2.4 OH.nm -2 was obtained and it is consistent with values generally reported in literature (between 2 and 5 OH.nm -2 ) [START_REF] Mueller | H surface density of SiO2 and TiO2 by thermogravimetric analysis[END_REF][START_REF] Zw | The adsorption and reaction of a titanate coupling reagent on the surfaces of different nanoparticles in supercritical CO 2[END_REF]. Surface of these SiO2 nanoparticles was then modified with various MPTS molar ratios (R = MPTS / SiO2 nanoparticle = 50, 75, 100 and 150 -from 0.48 to 1.44 mmol MPTS) in a water / methanol (5/20 (v/v)) mixture. The obtained colloidal solution was heated at reflux for 15h and the solution becomes cloudy. After methanol evaporation, a viscous white solution was obtained (Figure 1). The modified nanoparticles were dried at 90°C for 1 h in order to eliminate water and residual MPTS before analysis. FT-IR analysis shows that the C-H stretching vibrations of MPTS methoxy groups at 2841 cm -1 have disappeared proving that the hydrolysis of these functions is complete whatever the R ratio. In addition, the intensities of the specific bands of stretching modes of carbonyl C=O at 1717 cm -1 and of methacrylate C=C at 1637 cm -1 can be compared by normalizing the different spectra with the absorption band at 1100 cm -1 characteristic of Si-O-Si vibrations. Whatever the R values, the ratio of the C=O and C=C band areas is constant at about 0.2, the same value of that measured on polycondensed PMPTS.

These analyses show that the MPTS methacrylate function does not polymerize during the SiO2 nanoparticle modification. To confirm that MPTS is grafted to the nanoparticles surface as expected, both polymers have been synthetized from MPTS solution in the absence of nanoparticle and they have been characterized by TGA analysis. A first polymer (PMPTS) results

of the free-radical polymerization of the MPTS methacrylate functions initiated by DCPD. A second polymer (PolyMPTS) is obtained from the condensation of the MPTS silanol functions which occurs after the hydrolysis of methoxy group. A 5% weight loss is measured by TGA analysis at 230°C for PolyMPTS and at 175°C for PMPTS (Fig. 2.).

In addition, methoxy function are not hydrolysed in PMPTS and they can be thus degraded by the temperature increase: the residual weight is thus close to zero. In contrast, the MPTS silanol functions resulting of the hydrolysis of methoxy group are transformed in silica after polycondensation. This silica cannot be thermally degraded, and the residual weight differs from zero. The thermal behavior of these organic MPTSbased materials is significantly different of that synthesized in the presence of SiO2 nanoparticles which is stable up to 320°C (Fig. 2.). Thus, MPTS seems preferentially to condense on the nanoparticle surface rather than in homopolymer form in the solution under the used experimental conditions. Effect of MPTS / SiO2 nanoparticle molar ratio (R = 50, 75, 100 and 150) on the MPTS grafting ratio -defined as the ratio of grafted MPTS and MPTS used for the synthesis -was then evaluated (Table 1). The grafting ratio was determined from the weight loss (Supporting information -Fig. S1.) and calculated as [START_REF] Bartholome | Nitroxidemediated polymerization of styrene initiated from the surface of fumed silica. Comparison of two synthetic routes[END_REF] (Eq.3.): where WΔT is the weight loss at 750°C, M the molar weight of grafted-MPTS, Sspe the specific surface, and WSiO2 the weight loss of unmodified nanoparticles, respectively. However, in view of the introduced MPST amount, the MPTS grafting ratio decreases from 92 to 79% when R increases. These experimental values are close to the theoretical grafting ratios that shows that most OH functions on the nanoparticle surface have been modified by MPTS. From these measurements, it is also possible to estimate the molecular area of MPTS at the nanoparticle surface (Table 1), and to deduce the diameter of the MPTS's projection surface (assumed to a disk) which varies from 1.2 to 0.75 nm.

Second, for the material synthesis, the MPTS-modified SiO2 nanoparticles have been extracted from the aqueous solution with MMA. Whereas a sedimentation is always observed with unmodified nanoparticles, the extraction of modified nanoparticles with MMA is spontaneous and the resulting MMA solution is transparent. In order to verify the dispersion, a dynamic light scattering (DLS) analysis was performed on 1 wt% MPTS-SiO2 nanoparticles in MMA solution. The nanoparticle diameter is 7-10 nm whatever the R ratio (Table 1) while the initial size of nanoparticles is 4 nm. The increase would correspond to a MPTS shell of about 1.5 to 3 nm thick in agreement with the MPTS length estimated to 1.1 nm and in agreement with previous results reported in the literature [START_REF] Bauer | Preparation of Scratch-and Abrasion-Resistant Polymeric Nanocomposites by Monomer Grafting onto Nanoparticles[END_REF]. However, the MPTS's high is significantly higher that the diameter of the occupied surface by MPTS. Thus, it seems that MPST adopt rather a brush conformation at the surface of silica nanoparticles.

It was also checked by DLS analysis that the MPTS-SiO2 nanoparticles remain stable at 25°C in MMA at least 3 h, time required for the PMMA network synthesis. It is thus possible to control the size and dispersion of MPTS-SiO2 nanoparticles in MMA solution, a required condition for synthesis of transparent glass. For next work, silica nanoparticles were modified with a MPTS / SiO2 nanoparticles ratio of R = 100. Well dispersed MPTS-modified SiO2 particles (in black) were observed in the PMMA matrix. In addition, they are not aggregated since the nanoparticle sizes estimated from this image is in the range of 5.1 ± 0.7 nm (average on 50 measurements). This value is close to that of SiO2 particles (4.1 ± 1.3 nm and 6.7 ± 2 nm for unmodified and modified nanoparticles, respectively). Thus MPTS-modified SiO2 used as PMMA's crosslinker allows elaborating nanoscale dispersed hybrid materials.

Synthesis and Characterization of hybrid

Second, the methacrylate function number on nanoparticles surface that involves during MMA polymerization have been evaluated. To this end, the molecular weight between crosslinks (Mc) has been determined by swelling of the PMMAx-SiO2 networks with dichloromethane (Table 2). Indeed, dichloromethane is a PMMA good solvent since it has a solubility parameter (δCH2Cl2 = 20.2 MPA 1/2 ) close to that of PMMA (δPMMA = 19.4 MPA 1/2 ) [START_REF] Barton | Handbooks of solubility parameters and other cohesion parameters[END_REF]. The maximum uptake decreases from 320 to 270% when the SiO2 nanoparticle proportion increases from 4.5 to 7 wt%, with evidence of a crosslinking density increase. The theoretical weight Mc of PMMAx-SiO2 networks was d calculated with the Flory and Rehner equation [START_REF] Lange | Determination of the degree of swelling and crosslinking of extremely small polymer gel quantities by analytical ultracentrifugation[END_REF] (Eq.4.):

" # $ % & ' ( & / *( & $/+ , -. /*( & 0( & 01 $& ( & & Equation 4
where V1 is molar fraction of CH2Cl2 (63.9 L.mol -1 ) [START_REF] Hansen | Hansen Solubility Parameters: A user's Handbook[END_REF], ρ2 the PMMA density (1.18 g.cm -3 ), υ2 the polymer volume fraction, and χ12 the interaction parameter PMMA / CH2Cl2 set at 0.34 [START_REF] Hildebrand | The Solubility of Nonelectrolytes[END_REF].

x (wt%) 4. i.e. only about 10 mol% methacrylate functions on the nanoparticle surface would be involved in PMMA crosslinking. However, the Flory-Rehner relationship is valid for loosely cross-linked networks in which the size and thus volume fraction of the crosslinker is negligible. This is not the case with the networks containing multifunctional silica cross-linkers. Therefore, the data of experimental Mc and methacrylate functions taking part in the cross-linking process are most likely far from the real values, and thus, these data should be considered as rough estimates. Nevertheless, these results confirm that the nanoparticles act as PMMA's crosslinker.

The cross-linking density of PMMAx-SiO2 networks was then studied by DMA measurements. Indeed, higher the crosslinking density, higher the mechanical relaxation temperature of the material and higher the storage modulus at rubbery plateau [START_REF] Berrebi | Development of organic glass using Interpenetrating Polymer Networks with enhanced resistance towards scratches and solvents[END_REF]. Storage moduli (E') and loss factor (tanδ) of the PMMAx-SiO2 networks were reported versus as a function of temperature in Fig. 5. A main α-mechanical relaxation was detected whatever the nanoparticles amount. Its temperature (Tα) increases from 124 to 137° C and the tanδ peak intensity decreases of about 0.4 when the MPTS-SiO2 nanoparticles proportion increases from 4.5 to 13.5 wt% (Fig. 5A.). In addition, up to 8.5 wt%, the tanδ peak width becomes thinner that confirms evidence of a more homogeneous crosslinking density. Simultaneously, the storage modulus at the rubbery plateau (above 150°C) increases from 3 to 5 MPa when the amount of nanoparticles increases from 4.5 to 13.5 wt% (Fig. 5B.). These results highlights that both density and homogeneity of PMMA's crosslinking increase with the nanoparticle amount in the material.

A β-mechanical relaxation of PMMA related to the rotation side groups is also observed around Tβ=100°C on tanδ-temperature curves [START_REF] Fukao | Dynamics of α and β processes in thin polymer films: Poly(vinyl acetate) and poly(methyl methacrylate)[END_REF]. The corresponding tanδ peak (Fig. 5A) and storage modulus decrease (Fig. 5B) disappear when the amount of MPTS-SiO2 nanoparticles increases. Thus, the higher the crosslinking density, the lower freedom of movement of its side chains.

All of the results show that the modified silica nanoparticles behave as the PMMA's crosslinker.

Finally, the stability of PMMA5-SiO2 networks under artificial UV ageing was studied.

The UV-visible spectra were recorded at different ageing times and were compared with those recorded on PMMA network in which PMMA was cross-linked with organic EGDMA (Fig. 6). No spectral changes were observed for single PMMA network, whether MPTS-SiO2 nanoparticles are present. This confirms that the presence of nanoparticles does not affect the PMMA stability over time. Wavelength (nm) Wavelength (nm) Absorbance (AU) (AU)

(A) (B)
Thus, the synthesis of transparent PMMA networks can be carried out with silica nanoparticles functionalized with organosilane bearing methacrylate functions as cross-linker. These hybrid PMMAx-SiO2 networks have been then combined with a PCR39 ® polycarbonate network in an interpenetrating polymer network (IPN) architecture in order to obtain materials with a better abrasion resistance than single PMMA network or organic PMMA/PCR39 ® IPNs [START_REF] Berrebi | Development of organic glass using Interpenetrating Polymer Networks with enhanced resistance towards scratches and solvents[END_REF].

Synthesis and characterization of hybrid PMMAx-SiO2/PCR39 ® IPNs:

Hybrid PMMAx-SiO2/PCR39 ® IPNs were synthesized from a MMA solution containing MPTS-SiO2 nanoparticles in which CR39 ® , DCPD and BPO were added. As CR39 ® and MMA polymerize according to a free-radical mechanism. their polymerizations were initiated by two initiators decomposing at different temperatures, each being specific to a monomer, in order to limit their copolymerization and the resulting grafting of both networks. Thus DCPD, a low temperature initiator, can initiates the polymerization of reactive MMA at 35°C while a high temperature initiator, BPO (kd = 2.5 × 10 -5 s -1 ) initiates polymerization of the less reactive CR39 ® at 80°C [START_REF] Berrebi | Development of organic glass using Interpenetrating Polymer Networks with enhanced resistance towards scratches and solvents[END_REF]. The PCR39 ® weight proportion ranges from 25 to 75%. It does not need addition of a specific cross-linker because CR39 ® is tetrafunctional (its homopolymerization leads to a network formation). The thermal cure was set up to 3h at 35°C, 2h at 80°C and a post-cure for 1h at 100°C (Fig. against 85°C without nanoparticles. Similarly, the relaxation temperatures were detected at 108°C and 103°C for hybrid PMMA13-SiO2/PCR39 ® (25/75) IPNs containing or not nanoparticles, respectively. Simultaneously, the storage moduli at the rubbery plateau of hybrid PMMA13-SiO2/PCR39 ® IPNs are always slightly greater than those of the corresponding PMMAlinear/PCR39 ® semi-IPNs (Fig. 8 insert). In addition, the higher the PMMA proportion in IPNs, the more the differences of the modulus at the rubbery plateau and of the α-mechanical relaxation temperature between IPNs and semi-IPNs are significant. Therefore, it seems that the MPTS-SiO2 nanoparticles act mainly as the crosslinker of PMMA in these RIPs. To confirm this result, TEM images were recorded to locate nanoparticles (in PMMA or in PCR39 ® phase) (Fig. 4.). The images show clearly black and white domains. Due to the transmission difference, the clear domains have been assigned to an organic phase (PCR39 ® or / and PMMA) and the dark domains to the MPTS-SiO2 nanoparticles. The dispersion of nanoparticles in hybrid PMMA7-SiO2/PCR39 ® IPN containing 75 and 50 wt% PMMA (Fig. 4b and4c.), appears to be less homogeneous than in the hybrid PMMA7-SiO2 network (Fig. 4a).

The nanoparticles would be more concentrated in some organic domains than in others. In addition, the area of nanoparticle concentrated domains seems decrease with the PMMA amount in IPNs. Thus, the modified nanoparticles would be mainly located in the PMMA phase. Finally, the utilization of those nanoparticles would also an original method to stain one of the phases in an IPN architecture.

The last point of this study is the evaluation of scratch resistance of these IPNs (Fig. 9): the deeper the scratch, the lower the scratch resistance. As expected, the increase from 0 to 6 wt% MPTS-SiO2 nanoparticle content in the hybrid PMMAx-SiO2/PCR39 ® (50/50) IPN induces a decrease of the scratch depth from 3.7µm to around 1.5µm (Fig. 9A). Beyond 6 wt% MPTS-SiO2 nanoparticles content, the scratch depth remains the same. Thus, the scratch depths on the hybrid IPNs are twice lower than those carried out on the organic PMMAlinear/PCR39 ® IPNs. This result is in agreement with the fact that generally the addition of inorganic fillers addition significantly increases the material hardness which is directly related to anti-scratch properties [START_REF] Bauer | UV Curable Acrylate Nanocomposites: Properties and Applications[END_REF]. In order to identify which of PCR39 ® or SiO2-nanoparticles has an overriding effect on the scratch resistance, the scratch depth has also been measured as a function of the PCR39 ® proportion at 6 wt% nanoparticle proportion (with respect to PMMA). While the PCR39 ® proportion was varied from 25 to 75 wt%, the scratch depth seems constant around 0.8 µm (Fig. 9B). Thus, despite their dilution, the SiO2nanoparticle effect on the scratch resistance of the IPNs is greater than that of PCR39 ® .

Conclusion

While most of the hybrid interpenetrating polymer networks reported in literature are synthesized by elaborating the inorganic phase by an in-situ sol-gel reaction, we have shown that modified-SiO2 nanoparticles can be also directly included in PMMA phase and be evenly distributed inside. To this end, SiO2 nanoparticles must be previously modified with methacrylate functions which simultaneously allow a correct dispersion in MMA monomer and being the PMMA cross-linker. To assure material's transparency, silica less than 10 nm diameter has been chosen and previously

Fig. 1 .

 1 Fig. 1. Reaction pathway for (a) modification of SiO2 nanoparticles with 3-

Fig. 2 .

 2 Fig.2. Thermogravimetric analysis (TGA) of (--) MPTS, (---) PMPTS, (-⋅-) PolyMPTS

  PMMAx-SiO2 network: PMMAx-SiO2 hybrid networks have been synthesized by free-radical polymerization of MMA monomer in the presence of 0.5%wt DCPD initiator and different weight proportions of MPTS-SiO2 nanoparticles (2-13.5%) as crosslinker (Fig.1.). After mixture of precursors, a thermal program of 35°C for 3h and a post-cured at 80°C for 1h was applied. Transparent materials were obtained. First, the PMMA's crosslinking and the nanoparticles dispersion were analyzed. To show evidence of the cross-linking, the soluble fractions contained in the different single PMMAx-SiO2 hybrid networks have been quantified by Soxhlet extraction (Fig.3.).

Fig. 3 .

 3 Fig.3. Soluble fraction contained in PMMAx-SiO2 hybrid networks versus the weight

Fig. 4 .

 4 Fig.4. Transmission electron microscopy images of hybrid PMMA7-SiO2 network (X

Table 2 .

 2 Uptake in dichloromethane and experimental and theoretical molecular weights between crosslinks (Mc) of PMMAx-SiO2 networks. The estimated molecular weight between crosslinks (Mc) decreases from 2100 to 1500 when the nanoparticles proportion increases. Parallelly, the theoretical weight Mc has been calculated from the number of MPTS grafted on silica surface by assuming a homogeneous distribution in the material and a total polymerization of methacrylate functions. The theoretical Mc is about 10 times lower than the experimental Mc value,

Fig. 5 .

 5 Fig.5. (A) Loss factor and (B) Storage modulus as a function of temperature of PMMAx-

Fig. 6 .

 6 Fig.6. Absorbance of (A) organic PMMA5 network and (B) hybrid PMMA5-SiO2 network

  7.). All hybrid PMMAx-SiO2/PCR39 ® IPNs are transparent despite the presence of MPTS-SiO2 nanoparticles (the IPN's transmittance in the visible range is close to 90% except for PMMA13-SiO2/PCR39 ® (25/75) IPNclose to 80% -Supporting information -Fig.S2.).

Fig. 7 .

 7 Fig.7. Scheme of hybrid PMMAx-SiO2/PCR39 ® IPN synthesis.

Fig. 8 .

 8 Fig.8. Loss factor of hybrid PMMA13-SiO2/PCR39 ® IPNs (black line) and of

Fig. 9 .

 9 Fig.9. Scratch depth of hybrid PMMAx-SiO2/PCR39 ® IPNs as function of (A) MPTS-

  modified. First, single PMMA networks have been synthesized with various modified SiO2-nanoparticles amounts. The PMMA cross-linking has been checked by solidliquid extraction and DMA analysis. The dispersion of nanoparticles has been also checked by TEM microscopy and TGA analysis. These hybrid PMMA network have been then associated to polycarbonate network inside an IPN architecture. While IPNs are synthesized according to an in-situ synthesis, nanoparticles are mainly located in PMMA phase. As expected, transparency (transmittance higher than 80%) is kept and, finally, the addition of only 3 wt% of nanoparticles provides improved scratch resistance (hybrid IPN's scratch depth is twice lower than those of the simple PMMA network and of the organic PMMA/PCR39 ® IPN). This study has therefore opened a new way for the preparation of IPNs hybrid. It is possible to consider changing the nature of nanoparticles to provide additional properties to the materials as photo-luminescency.

Fig. S2 .

 S2 Fig.S2. Transmittance of hybrid PMMA13-SiO2 /PCR39 ® (a) (25/75), (b) (50-50) and (c)

  Since WSiO2 is very low (less than 2%), WΔT can be considered as coming only from the thermal degradation of the grafted MPTS on the nanoparticles.

	MPTS / SiO2 nanoparticle (R)	50	75	100	150
	MPTS grafting ratio (%)	92	91	83	79
	Theorical grafting degree (%)*	100	100	100	80
	Molecular area by MPTS (nm²)	1.15	0.77	0.64	0.45
	Diameter of MPTS** (nm)	1.21	0.99	0.90	0.75
	Modified nanoparticle diameter (nm)	8.7±2.4	10±2.4	6.7±2	10.1±2.4

* Calculated assuming a density of 120 OH by nanoparticle -** Assuming a circle of projection

Table

1

. Characteristic of SiO2 nanoparticles modified with different R molar ratios WΔT increases from 12 to 26% when the R molar ratio increases from 50 to 150.

Acknowledgements

We gratefully thank the Fondation des Sciences du Patrimoine for financing Mickaël Berrebi 's research grant (ANR-10-LABX-0094-01 -Reference: EUR-17-EURE-0021).

Data availability:

The raw/processed data required to reproduce these findings cannot be shared at this time due to technical or time limitations.