Rémi G Tilkin
email: remitilkin@gmail.com

Julien G Mahy
email: julien.mahy@uliege.be

Ana P F Monteiro

Artium Belet

Julio Feijóo

Mathilde Laird

Carole Carcel

Nicolas Régibeau

Bart Goderis

Christian Grandfils

Michel Wong

Chi Man

Stéphanie D Lambert

Protein encapsulation in mesoporous silica: influence of the mesostructured and pore wall properties

Keywords: biomaterials, bone reconstruction, protein encapsulation, silica gel, sol-gel process, structured silica, ordered mesoporous silica, SBA-15

h. Afterwards, the STI release slowed down and seemed to reach a plateau. On the opposite, the structured sample showed a steady release over time. Finally, the sample synthesized with BTEB did not show a significant release over the same period of time. This led us to the conclusion that the hydrophobicity of the silica surface plays a major role on the protein encapsulation and its release rate.

ABSTRACT

The goal of this work is to study the influence of the structured mesoporous silica (SMS), used as scaffolds for tissue engineering, on the encapsulation and on the release of a model bone morphogenetic proteins (BMP), i.e. Soybean Trypsin Inhibitor (STI).

First, SBA-15 silica samples were synthesized using tetraethyl orthosilicate (TEOS) as silica precursor in the presence of P123 as the surfactant in an acidic medium. Three homemade highpressure stainless steel reactors (with smaller Teflon reactors inside), each equipped with a temperature probe, were used for the syntheses. In addition, a swelling agent was added to increase the pore size. Indeed, the common pore size for those mesostructured materials lies between 6 nm and 8 nm, while the STI has an average radius of 4 nm. The reagent addition sequence was also investigated, by playing on the time of addition of the swelling agent. It was observed that when the swelling agent was added with TEOS, an ordered mesoporous silica was produced. On the contrary, if the swelling agent was added during the surfactant dissolution step, it resulted in an unstructured yet still mesoporous silica.

Because of the high affinity of STI for hydrophobic surfaces, SMS were also produced using silica precursors containing phenylene bridging groups (1,4-bis(triethoxysilyl)benzene, BTEB).

The BTEB samples exhibited two 2D-hexagonal structures with different wall thicknesses.

Synthesized materials were further characterized by infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), nitrogen sorption at 77 K, transmission electron microscopy (TEM), and small-angle X-ray scattering (SAXS).

The influence of both the silica composition and structure on STI encapsulation/release was studied. For samples synthesized with TEOS, the unstructured sample showed a higher protein loading and a higher protein release, which could be explained by a difference in the pore interconnectivity within the sample. In fact, a fast release of STI was observed during the first 24 1. INTRODUCTION Bone repair represents a flourishing topic. Every year, over 4 million surgical operations involving bone grafting or bone graft substitutes are performed worldwide [START_REF] Brydone | Bone grafting, orthopaedic biomaterials, and the clinical need for bone engineering[END_REF]. Bone represents the second most grafted organ after blood transfusion [START_REF] Campana | Bone substitutes in orthopaedic surgery: from basic science to clinical practice[END_REF]. Moreover, the importance of bone repair is expected to increase continuously in the next decades with the population aging (i.e. almost doubling of the population over 65 years in 2050) and the constant increase of need for healthcare worldwide [START_REF]Statistical abstracts of the United States: 2012 -Section 1. Population[END_REF].

Several methods have been proposed to recover the complete structure and function of the native bone, including the sustained and local administration of therapeutic biomolecules [START_REF] Bhattacharyya | Polymer-coated mesoporous silica nanoparticles for the controlled release of macromolecules[END_REF][START_REF] Chen | BMP-2-loaded silica nanotube fibrous meshes for bone generation[END_REF][START_REF] Tilkin | Protein encapsulation in functionalized sol-gel silica : Effect of the encapsulation method on the release kinetics and the activity[END_REF][START_REF] Tilkin | Protein encapsulation in functionalized sol -gel silica : influence of organosilanes and main silica precursors[END_REF]. Among these molecules, bone morphogenetic proteins (BMP), a family of cytokines, have been widely investigated [START_REF] Tilkin | Protein encapsulation in functionalized sol-gel silica : Effect of the encapsulation method on the release kinetics and the activity[END_REF][START_REF] Tilkin | Protein encapsulation in functionalized sol -gel silica : influence of organosilanes and main silica precursors[END_REF][START_REF] Lo | Studies of bone morphogenetic proteinbased surgical repair[END_REF][START_REF] Hwang | Immunogenicity of bone morphogenetic proteins[END_REF][START_REF] Bessa | Bone morphogenetic proteins in tissue engineering: the road from laboratory to clinic , part II (BMP delivery)[END_REF]. BMP are known to regulate the bone formation via the control of cell adhesion, proliferation, differentiation, and apoptosis. Studies have also demonstrated that the protein BMP-2 could promote the fracture repair by recruiting osteogenic progenitor cells and stimulating the bone differentiation of stem cells [START_REF] Kimura | Controlled Release of Bone Morphogenetic Protein-2 Enhances Recruitment of Osteogenic Progenitor Cells for De Novo Generation of Bone Tissue[END_REF][START_REF] Yamashita | The use of dynamic mechanical analysis to assess the viscoelastic properties of human cortical bone[END_REF][START_REF] Sun | Role of bone morphogenetic protein-2 in osteogenic differentiation of mesenchymal stem cells[END_REF]. Nevertheless, BMP cannot be directly injected into the fracture site because of their rapid clearance [START_REF] Bialy | Formulation, Delivery and Stability of Bone Morphogenetic Proteins for Effective Bone Regeneration[END_REF]. Moreover, an excessively high dose could lead to an undesirable growth of tumors or neovascularization of non-targeted tissues [START_REF] Jung | Highly aligned porous Ti scaffold coated with bone morphogenetic protein-loaded silica / chitosan hybrid for enhanced bone regeneration[END_REF]. Delivery systems with specific textural and chemical properties must therefore be designed to obtain a sustained delivery of BMP [START_REF] Schrier | Recombinant Human Bone Morphogenetic Protein-2 Binding and Incorporation in PLGA Microsphere Delivery Systems[END_REF][START_REF] Johnson | Sustained release of BMP-2 in a lipid-based microtube vehicle[END_REF][START_REF] Li | Surface Immobilization of TiO2 Nanotubes with Bone Morphogenetic Protein-2 Synergistically Enhances Initial Preosteoblast Adhesion and Osseointegration[END_REF]. In this study, a model protein (i.e. Soybean Trypsin Inhibitor, STI) was selected based on its properties (size, molecular weight, point of zero charge, and release kinetics, Table 1), which are similar to the BMP ones [START_REF] Hum | Bioactive glasses as carriers for bioactive molecules and therapeutic drugs : a review[END_REF][START_REF] Reiner | Protein incorporation within Ti scaffold for bone ingrowth using Sol-gel SiO2 as a slow release carrier[END_REF][START_REF] Agrawal | Protein release kinetics of a biodegradable implant for fracture[END_REF]. Moreover, this protein has been previously used to model BMP release from porous silica [START_REF] Tilkin | Protein encapsulation in functionalized sol-gel silica : Effect of the encapsulation method on the release kinetics and the activity[END_REF][START_REF] Reiner | Protein incorporation within Ti scaffold for bone ingrowth using Sol-gel SiO2 as a slow release carrier[END_REF][START_REF] Agrawal | Protein release kinetics of a biodegradable implant for fracture[END_REF][START_REF] Santos | Sol-gel derived carrier for the controlled release of proteins[END_REF]. 21,000 [START_REF] Reiner | Protein incorporation within Ti scaffold for bone ingrowth using Sol-gel SiO2 as a slow release carrier[END_REF][START_REF] Santos | Sol-gel derived carrier for the controlled release of proteins[END_REF] 26,000 [START_REF] Reiner | Protein incorporation within Ti scaffold for bone ingrowth using Sol-gel SiO2 as a slow release carrier[END_REF] Size (nm) 4.5 x 4.2 x 4 [START_REF] Song | Kunitz-type Soybean Trypsin Inhibitor Revisited : Refined Structure of its Complex with Porcine Trypsin Reveals an Insight into the Interaction Between a Homologous Inhibitor from Erythrina caffra and Tissue-type Plasminogen Activator[END_REF] 7 x 3.5 x 3 [START_REF] Scheufler | Crystal structure of human bone morphogenetic protein-2 at 2.7 Å resolution1[END_REF] Point of zero charge (pH unit) 4.5 [START_REF] Chang | Selective Isolation of Trypsin Inhibitor and Lectin from Soybean Whey by Chitosan / Tripolyphosphate / Genipin Co-Crosslinked Beads[END_REF] 4.8-5.1 [START_REF] Yang | Bone morphogenetic proteins: Relationship between molecular structure and their osteogenic activity[END_REF] Structured mesoporous silica (SMS) with well-defined properties (e.g. ordered pores, controlled pore size distributions), are attractive to finely tune the loading and the release rate of biopharmaceutical drugs [START_REF] Vallet-Regi | A New Property of MCM-41 : Drug Delivery System[END_REF][START_REF] Fernanandez-Nunez | Ibuprofen Loading in Surfactant-Templated Silica : Role of the Solvent According to the Polarizable Continuum Model[END_REF][START_REF] Zelenak | Applied Surface Science Ordered cubic nanoporous silica support MCM-48 for delivery of poorly soluble drug indomethacin[END_REF][START_REF] Szewczyk | Amino-modified mesoporous silica SBA-15 as bifunctional drug delivery system for cefazolin : Release profile and mineralization potential[END_REF]. Moreover, thanks to the well-defined periodic geometry of SMS, the protein release can be further regulated by grafting specific responsive molecules, called gates or caps, at the pore opening after loading the biomolecule [START_REF] Mekaru | Development of mesoporous silica-based nanoparticles with controlled release capability for cancer therapy ☆ a ) Liposome[END_REF][START_REF] Narayan | Mesoporous Silica Nanoparticles : A Comprehensive Review on Synthesis and Recent Advances[END_REF][START_REF] Wang | Microporous and Mesoporous Materials Ordered mesoporous materials for drug delivery[END_REF][START_REF] Ukmar | Ordered mesoporous silicates as matrices for controlled release of drugs[END_REF]. These gates could indeed prevent the release of the cargo. Once inside the body, the opening of these gates can be triggered by a specific stimulus (e.g. a change in pH or in temperature) to release the loaded biomolecules [START_REF] Théron | Hybrid Mesoporous Silica Nanoparticles with pH-Operated and Complementary H-Bonding Caps as an Autonomous Drug-Delivery System[END_REF][START_REF] Théron | pH-operated hybrid silica nanoparticles with multiple H-bond stoppers for colon cancer therapy[END_REF][START_REF] Song | pH-Controllable drug release using hydrogel encapsulated mesoporous silica[END_REF][START_REF] Gan | A dual-delivery system of pHresponsive chitosan-functionalized mesoporous silica nanoparticles bearing BMP-2 and dexamethasone for enhanced bone regeneration[END_REF][START_REF] Baeza | Magnetically Triggered Multidrug Release by Hybrid Mesoporous Silica Nanoparticles[END_REF][START_REF] Fu | Control of Molecular Transport Through Stimuli-Responsive Ordered Mesoporous Materials[END_REF][START_REF] Yu | Improved controlled release of protein from expanded-pore mesoporous silica nanoparticles modified with cofunctionalized poly(n-isopropylacrylamide) and poly(ethylene glycol) (PNIPAM-PEG)[END_REF]. To our knowledge, Ehlert et al. are the only ones who have compared the encapsulation of BMP-2 in structured and unstructured silica using an impregnation technique [START_REF] Ehlert | Amino-modified silica surfaces efficiently immobilize bone morphogenetic protein 2 ( BMP2 ) for medical purposes[END_REF]. Surprisingly, the authors have not detected BMP-2 being encapsulated neither in the ordered nor in the disordered silica materials. These observations are therefore in contrast with previously findings published in the literature [START_REF] Tilkin | Protein encapsulation in functionalized sol-gel silica : Effect of the encapsulation method on the release kinetics and the activity[END_REF][START_REF] Tilkin | Protein encapsulation in functionalized sol -gel silica : influence of organosilanes and main silica precursors[END_REF][START_REF] Reiner | Protein incorporation within Ti scaffold for bone ingrowth using Sol-gel SiO2 as a slow release carrier[END_REF][START_REF] Santos | Sol-gel derived carrier for the controlled release of proteins[END_REF][START_REF] Tang | Bioinspired trimodal macro / micro / nano-porous scaffolds loading rhBMP-2 for complete regeneration of critical size bone defect[END_REF][START_REF] Nicoll | In vitro release kinetics of biologically active transforming growth factor-β1 from a novel porous glass carrier[END_REF].

Considering SMS advantages, we envisaged the synthesis of SBA-15 type silica with larger mesopores. In addition to its stability and absence of toxicity, SBA-15 was chosen for its high adsorption capacity [START_REF] Tan | Preparation and physical characterization of calcium sulfate cement / silica-based mesoporous material composites for controlled release of BMP-2[END_REF][START_REF] Garcia | Microporous and Mesoporous Materials Protein encapsulation in SBA-15 with expanded pores[END_REF][START_REF] Katiyar | Synthesis of ordered large pore SBA-15 spherical particles for adsorption of biomolecules[END_REF][START_REF] Diao | Effect of Pore-size of Mesoporous SBA-15 on Adsorption of Bovine Serum Albumin and Lysozyme Protein *[END_REF][START_REF] Yiu | Size selective protein adsorption on thiol-functionalised SBA-15 mesoporous molecular sieve[END_REF]. The synthesis of SMS was investigated through the use of TEOS as silica precursor [START_REF] Narayan | Mesoporous Silica Nanoparticles : A Comprehensive Review on Synthesis and Recent Advances[END_REF][START_REF] De | Block copolymer-templated mesoporous oxides[END_REF][START_REF] Pagar | Mesoporous Silica : A Review[END_REF][52][START_REF] Zhao | Recent advances of mesoporous materials in sample preparation[END_REF]. To obtain larger mesopores than traditional SBA-15, swelling agents such as alcohols, aliphatic/aromatic hydrocarbons are typically added during the synthesis [START_REF] Cao | Synthesis of Ultra-Large-Pore SBA-15 Silica with Two-Dimensional Hexagonal Structure Using Triisopropylbenzene As Micelle Expander[END_REF][START_REF] Huang | Journal of Colloid and Interface Science Synthesis of ultra-large-pore FDU-12 silica using ethylbenzene as micelle expander[END_REF][START_REF] Zhang | Engineered Complex Emulsion System : Toward Modulating the Pore Length and Morphological Architecture of Mesoporous Silicas[END_REF][START_REF] Johansson | Microporous and Mesoporous Materials The effects on pore size and particle morphology of heptane additions to the synthesis of mesoporous silica SBA-15[END_REF][START_REF] Kruk | Access to Ultralarge-Pore Ordered Mesoporous Materials through Selection of Surfactant / Swelling-Agent Micellar Templates[END_REF][START_REF] Deodhar | Controlled release and intracellular protein delivery from mesoporous silica nanoparticles[END_REF]. These molecules dissolve inside the hydrophobic part of the micelles, causing a swelling of the later and a subsequent increase in pore size of the final material. Nevertheless, the presence of these additives are known to alter the organization of the material, notably causing a reduction of the degree of organization of the material [START_REF] Garcia | Microporous and Mesoporous Materials Protein encapsulation in SBA-15 with expanded pores[END_REF][START_REF] Kruk | Access to Ultralarge-Pore Ordered Mesoporous Materials through Selection of Surfactant / Swelling-Agent Micellar Templates[END_REF]. When using surfactants with a low hydrophilic-lipophilic balance, such as the Pluronic P123, the addition of a swelling agent with low to moderate solubility in water has been recommended to produce SMS combining welldefined structures and large pores [START_REF] Kruk | Access to Ultralarge-Pore Ordered Mesoporous Materials through Selection of Surfactant / Swelling-Agent Micellar Templates[END_REF]. 1,3,5-triisopropylbenzene (TIPB) is therefore chosen, based on its low water-solubility [START_REF] Garcia | Microporous and Mesoporous Materials Protein encapsulation in SBA-15 with expanded pores[END_REF][START_REF] Cao | Synthesis of Ultra-Large-Pore SBA-15 Silica with Two-Dimensional Hexagonal Structure Using Triisopropylbenzene As Micelle Expander[END_REF][START_REF] Kruk | Access to Ultralarge-Pore Ordered Mesoporous Materials through Selection of Surfactant / Swelling-Agent Micellar Templates[END_REF].

In a second step, based on the high affinity of STI for hydrophobic surfaces [START_REF] Tilkin | Protein encapsulation in functionalized sol -gel silica : influence of organosilanes and main silica precursors[END_REF], SMS were also synthesized with 1,4-bis(triethoxysilyl)benzene (BTEB). This silica precursor contains a phenylene bridging group between two alkoxysilane moieties (Figure 1), which ensures the regular distribution of these groups within the organosilica walls [START_REF] Corriu | New Mixed Organic-Inorganic Polymers: Hydrolysis and Polycondensation of Bis(trimethoxysilyl) organometallic Precursors[END_REF]. The packing of the phenylene groups inside the walls reduces their reactivity and accessibility compared to pendant groups [START_REF] Tilkin | Protein encapsulation in functionalized sol -gel silica : influence of organosilanes and main silica precursors[END_REF][START_REF] Muramoto | Preparation of Periodic Mesoporous Organosilica with Large Mesopores Using Silica Colloidal Crystals as Templates Received[END_REF]. The interactions between the phenylene groups and the protein are therefore weakened thus modulating the STI adsorption strength. The materials were further characterized by infrared spectroscopy (FTIR) to study their chemical composition, thermogravimetric analysis (TGA) to determine the organic content, as well as nitrogen adsorption-desorption at 77 K, transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) to assess their textural and morphological properties. The kinetics of STI release were determined over a period of 6 weeks. In this section, the activity of the proteins is expressed in N α -Benzoyl-L-arginine ethyl ester hydrochloride (BAEE) units. One BAEE unit yields a difference in the absorbance at 253 nm

MATERIALS AND METHODS

Materials

BTEB synthesis

This procedure was adapted from Birault et al. [START_REF] Birault | Synthesis of lamellar mesostructured phenylene-bridged periodic mesoporous organosilicas ( PMO ) templated by polyion complex ( PIC ) micelles[END_REF]. BTEB is prepared by Barbier-Grignard reactions from the respective aryl dibromide and TEOS, as shown in Equation 1.

(

) 1
The reaction was carried out under argon by use of a vacuum line and Schlenk tube techniques.

Solvents were dried and distilled before use. To a solution of magnesium turnings (15 g, 0.62 mol) in tetrahydrofuran (THF, 100 mL) placed under argon in a three-neck flask equipped with a magnetic stirrer, a condenser, and an addition funnel, 1,2-dibromoethane (5 mL) was added dropwise to activate magnesium. When the bubbles from the magnesium activation have stopped, TEOS (450 mL, 2.0 mol) and THF (200 mL) were added. Then, ). 29 Si NMR (80 MHz, CDCl3, δ, ppm): -58.10.

Sample preparation

Two silica precursors were used for the synthesis of SMS, i.e. TEOS and BTEB. The samples synthesized with TEOS were denoted TEOS-X with X corresponding to the sample number, while the sample produced using BTEB was denoted BTEB.

Synthesis using TEOS as silica precursor

This procedure was adapted from Cao et al. [START_REF] Cao | Synthesis of Ultra-Large-Pore SBA-15 Silica with Two-Dimensional Hexagonal Structure Using Triisopropylbenzene As Micelle Expander[END_REF]. Triblock copolymer Pluronic P123 was selected as template and 1,3,5-triisopropylbenzene (TIPB) as swelling agent. The synthesis conditions were selected in order to provide a molar ratio TEOS:P123:TIPB:NH For calcination, the samples were heated up to 550 °C at a rate of 1 °C/min for 6 h, under an air flux. For the acidic treatment, untreated silica (0.5 g) was placed in a solution containing ethanol (143 mL) and HCl (4.3 mL, 37 %) for 72 h under reflux. The materials were recovered by centrifugation, washed with ethanol, and dried at 80 °C under vacuum for 24 h. This treatment was applied once for the TEOS-2 sample and twice for the TEOS-3 sample.

Table 2 -Synthesis conditions of the samples synthesized using TEOS.

Sample Removal treatment TIPB addition during TEOS-1

Calcination TEOS addition TEOS-2

Acidic x1 TEOS addition TEOS-3

Acidic x2 TEOS addition

TEOS-4

Calcination P123 and NH 4 F dissolution

Synthesis using BTEB as silica precursor

This procedure was adapted from Laird et al. [START_REF] Laird | Single-template periodic mesoporous organosilica with organized bimodal mesoporosity[END_REF]. Triblock copolymer Pluronic P123 was selected as template. The synthesis conditions were selected in order to provide a molar ratio BTEB:P123:HCl:H 2 O of 1:0.034:0.462:402.

P123 (1.47 g) was dissolved in deionized water (53.6 mL) at 35 °C. HCl (310 µL, 37 %) was added and the resulting mixture was cooled to 0 °C. BTEB (1.5 g) was then added dropwise and the solution was stirred at 0 °C for 1 h. The solution was then heated to 35 °C for 24 h and then to 100 °C for 24 h under continuous stirring. The material was recovered by centrifugation and washed with deionized water. Surfactant was removed via acidic treatment. The hybrid silica (0.5 g) was extracted in a solution containing HCl (4.3 mL, 37 %) in ethanol (143 mL) for 72 h under reflux. The silica material was recovered by centrifugation, washed with ethanol, and dried at 80

°C under vacuum for 24 h.

Protein encapsulation

The proteins were encapsulated via an impregnation method (performed in triplicates). The samples were impregnated in a STI solution (1 mg/mL in phosphate buffer saline, PBS: 1.4 mM KH 2 PO 4 , 10 mM Na 2 HPO 4 , 137 mM NaCl, and 2.7 mM KCl, pH = 7.4) for 72 h at 25 °C under stirring (20 mg of sample per mL of solution). The suspensions were then centrifuged at 10,000 rpm for 5 min and the supernatant was removed. The particles were finally freeze-dried for 72 h.

Sample characterization 2.4.1. Physicochemical characterization

The purity of the synthesized organosilane was assessed by nuclear magnetic resonance (NMR).

Liquid 1 H and 29 Si NMR spectra were recorded on a Bruker Avance 400 MHz spectrometer in CDCl 3 at room temperature and at concentrations of around 1 and 10 mg/mL respectively. All 29 Si NMR spectra were proton decoupled.

The physicochemical characterization of the TEOS and BTEB samples was performed on the samples before protein encapsulation.

The presence of specific functional groups was confirmed by Fourier transformed infrared (FTIR) spectroscopy in ATR mode using an IRAffinity-1 from Shimadzu. The following instrumental settings were used: absorbance, range from 4000 to 400 cm -1 , 30 scans, resolution of 2 cm -1 . The spectra were normalized to the peak corresponding to Si-O-Si stretching (around 1050-1060 cm -1 ).

The organic content was determined by thermogravimetric analysis (TGA, TGA 7 from Perkin-Elmer) in platinum crucibles under air atmosphere (flow rate of 58 cm³/min) with a heating rate of 20 °C/min from 50 °C to 90 °C and 10 °C/min from 90 °C to 900 °C. The weight loss fraction w (i.e. weight loss (g) divided by the sample weight (g)) was measured between 150 °C and 900 °C.

The surface concentration of -OH groups (OH, -OH group/nm²) was calculated via Equation 2(2

)
where w is the weight loss fraction (-), MM H2O is the molar mass of water (18.02 g/mol), S BET is the specific surface area evaluated using Brunauer, Emmett, and Teller theory (m²/g), and N A is the Avogadro's number (6.022 x 10 23 particle/mol).

The textural and morphological properties were characterized by nitrogen adsorption-desorption isotherms at 77 K in an ASAP 2420 multi-sampler adsorption-desorption volumetric device from Micromeritics. From these isotherms, the total pore volume (V P ) was defined as the specific liquid volume adsorbed at the saturation pressure of nitrogen, the specific surface area was evaluated using Brunauer, Emmett, and Teller theory (S BET ), and the mesoporous size distribution was determined by Barrett, Joyner, and Halenda theory (d BJH ) [START_REF] Lecloux | Texture of Catalysts[END_REF][START_REF] Pirard | Development by the sol-gel process of highly dispersed Ni-Cu/SiO2 xerogel catalysts for selective 1,2dichloroethane hydrodechlorination into ethylene[END_REF].

The textural properties were also studied by small-angle X-ray scattering (SAXS) using an

XeuSS setup form Xenocs. The X-ray source was a molybdenum anode, delivering a high-energy monochromatic beam (λ = 0.7107 Å) and providing structural information over scattering vectors q (with q = (4sin)/ and  being half the scattering angle) ranging from 0.02 to 2.3 Å -1 . Only the range 0.02 < q < 0.2 Å -1 was ultimately considered for analysis. The flight tube between sample and the MAR345 image plate detector was purged with helium to avoid air scattering.

The data acquisition time was set to 1800 s. The samples were placed in closed aluminum DSC pan. A conversion from the MAR detector data file format to a 16 bits .tiff file was made using FIT2D free software from ESRF (France). An azimuthal averaging, taking into account the sample-detector distance and detector tilting angle, was performed via the free Matlab ConeX software, developed by Gommes and Goderis [START_REF] Gommes | CONEX , a program for angular calibration and averaging of two-dimensional powder scattering patterns[END_REF], and using silver behenate as a calibration

where a xxx is the lattice parameter corresponding to the (xxx) reflection (Å) and q xxx is the scattering vector corresponding to the (xxx) reflection (Å -1 ) [START_REF] Zienkiewicz-Strzałka | Small-angle X-ray scattering (SAXS) studies of the structure of mesoporous silicas[END_REF]. The wall thickness was determined via Equation 6 [START_REF] Galarneau | True Microporosity and Surface Area of Mesoporous SBA-15 Silicas as a Function of Synthesis Temperature[END_REF]. It is important to notice that, as the pore are cylindrical, the wall thickness is not constant and that the value determined here is an average value.

(

) 6
The specific mesoporous volume (V meso ), the specific microporous volume (V micro ), and the specific mesoporous surface (S meso ) were evaluated based on the nitrogen adsorption-desorption and SAXS measurements. For the structured samples (i.e. TEOS-1, TEOS-2, and BTEB), the specific mesoporous volume (V meso ) was determined using Equation 7, the specific microporous volume (V micro ) was evaluated via Equation 8, and the specific mesoporous surface was calculated using Equation 8 [START_REF] Galarneau | True Microporosity and Surface Area of Mesoporous SBA-15 Silicas as a Function of Synthesis Temperature[END_REF].

(

) 7
where ρ Si is the density of amorphous silica (i.e. 2.2 g/cm³)

For the unstructured sample (i.e. TEOS-4), the specific microporous volume (V micro ) was evaluated via Dubinin-Radushkevich theory [START_REF] Pirard | Development by the sol-gel process of highly dispersed Ni-Cu/SiO2 xerogel catalysts for selective 1,2dichloroethane hydrodechlorination into ethylene[END_REF], and the specific mesoporous volume (V meso )

was determined using Equation 9.

(

) 9
The textural properties were finally assessed via transmission electron microscopy (TEM). The samples were embedded in epoxy resin SPI-PON 812 (hard mixture, SPI-CHEM, 02659-AB) and left to polymerize for 3 days at 60 °C. Ultrathin sections (thickness = 70-80 nm) were obtained using a diamond knife on an ultramicrotome Reichert Ultracut E and observed in a TEM/STEM Tecnai G2 twin working at 200 kV accelerating voltage and spotsize 3. The lattice parameter (a TEM ) and the Fast-Fourier transform (FFT) analyses of selected regions of the TEM images were obtained using ImageJ software (Version 1.53a). The wall thickness was determined via Equation 6.

Biochemical characterization

Protein loading within silica was evaluated by measuring the STI concentration remaining in the supernatant after 72 h of incubation via the Lowry method using STI as a standard (absorbance measurement at 750 nm, spectrophotometer Lambda 25 from Perkin Elmer) [START_REF] Lowry | Protein measurement with the folin phenol reagent[END_REF].

The kinetics of protein release was determined by immersing 20 mg of sample in 1 mL of maleic acid buffer (100 mmol/L, pH = 7.4) in closed vials. This analysis was performed in triplicates.

Materials were incubated at 37 °C under continuous agitation. At scheduled times (i.e. 30 min, 3 h, 6 h, 1 day, 2 days, 3 days, 1 week, 2 weeks, 4 weeks, 6 weeks), the samples were centrifuged at 10,000 rpm for 5 min. The supernatant was removed, stored at 4 °C until analysis, and replaced by fresh maleic acid buffer. The STI concentration was assessed via the Lowry method using STI as standard (absorbance measurement at 750 nm, spectrophotometer Lambda 25 from Perkin Elmer) [START_REF] Lowry | Protein measurement with the folin phenol reagent[END_REF].

RESULTS AND DISCUSSION

Chemical composition

The effect of the surfactant removal treatment is investigated through two processes: calcination and acidic treatment. Calcination is performed to burn out the surfactant [START_REF] Huang | Calcining silica gels at different drying stages[END_REF][START_REF] Davis | Pore structure evolution in silica gel during aging / drying I . Temporal and thermal aging[END_REF]. This technique is simpler and faster than other techniques and can be easily implemented at an industrial scale.

Moreover, this procedure allows the total removal of residual solvent or unreacted species.

Unfortunately, this approach sometimes leads to a considerable shrinkage of the pores and thus a decrease in the pore size. For obvious reasons, the calcination is not suitable in the case of silica containing organic compounds, as it would damage the organic moieties. To tackle these problems, acidic treatment is a suitable alternative. Indeed, it consists in stirring the material in an ethanol solution containing hydrochloric acid at 80 °C for 3-5 days [START_REF] Davis | Pore structure evolution in silica gel during aging / drying I . Temporal and thermal aging[END_REF][START_REF] Davis | Pore structure evolution in silica gel during aging / drying II . Effect of pore fluids[END_REF][START_REF] Wu | Synthesis of mesoporous silica nanoparticles[END_REF]. Acidic treatment has been widely used despite its longer processing time compared to calcination because it allows to maintain the integrity of the pore size distribution while efficiently removing the surfactant molecules. This strategy prevents the pore shrinkage phenomenon associated with calcination.

Being performed at a lower temperature (typically below 100 °C), acidic treatment can also be applied to silica containing organic fragments, as the latter are not degraded during the process.

The FTIR absorption spectra of the TEOS samples are displayed in Figure 2 (a-c). Due to the similarities between the spectra of the TEOS-1 and TEOS-4 samples, only the spectrum of the TEOS-1 sample is presented. All FTIR spectra are available in Supplementary Materials, Figure S1. The four FTIR spectra exhibit the characteristic peaks of silica. The peaks corresponding to the bending of Si-O-Si groups are observed at 440-443 cm -1 [START_REF] Mansur | FTIR and UV-vis study of chemically engineered biomaterial surfaces for protein immobilization[END_REF][START_REF] Péré | Quantitative assessment of organic compounds adsorbed on silica gel by FTIR and UV-VIS spectroscopies: the contribution of diffuse reflectance spectroscopy[END_REF][START_REF] Antony | Highly dispersed Cu(II), Co(II) and Ni(II) catalysts covalently immobilized on imine-modified silica for cyclohexane oxidation with hydrogen peroxide[END_REF]. The stretching of Si-O-Si groups is detected at 796 and 811 cm -1 (i.e. symmetric stretching), at 1057-1067 cm -1 (i.e.

transversal optic mode of asymmetric stretching), and in the 1110-1300 cm -1 (i.e. longitudinal optic mode of asymmetric stretching) [START_REF] Péré | Quantitative assessment of organic compounds adsorbed on silica gel by FTIR and UV-VIS spectroscopies: the contribution of diffuse reflectance spectroscopy[END_REF][START_REF] Antony | Highly dispersed Cu(II), Co(II) and Ni(II) catalysts covalently immobilized on imine-modified silica for cyclohexane oxidation with hydrogen peroxide[END_REF][START_REF] Capeletti | Infrared and Raman spectroscopic characterization of some organic substituted hybrid silicas[END_REF][START_REF] Osswald | FTIR spectroscopic study on liquid silica solutions and nanoscale particle size determination[END_REF]. Characteristics peaks of silanol groups are found in the region 540-560 cm -1 (i.e. rocking mode of Si-OH groups) and around 950-985 cm -1 (i.e. asymmetric stretching of Si-OH groups) [START_REF] Mansur | FTIR and UV-vis study of chemically engineered biomaterial surfaces for protein immobilization[END_REF][START_REF] Antony | Highly dispersed Cu(II), Co(II) and Ni(II) catalysts covalently immobilized on imine-modified silica for cyclohexane oxidation with hydrogen peroxide[END_REF][START_REF] Capeletti | Infrared and Raman spectroscopic characterization of some organic substituted hybrid silicas[END_REF][START_REF] Osswald | FTIR spectroscopic study on liquid silica solutions and nanoscale particle size determination[END_REF]. These bands are weaker in the case of the calcined sample compared to the untreated sample and the sample after acidic treatment. This can be explained by the condensation of adjacent silanol groups into siloxane bonds during the calcination [START_REF] Warring | Sur fi cial Siloxane-to-Silanol Interconversion during Room-Temperature Hydration/Dehydration of Amorphous Silica Films Observed by ATR-IR and TIR-Raman Spectroscopy[END_REF][START_REF] Etienne | Analytical investigation of the chemical reactivity and stability of aminopropyl-grafted silica in aqueous medium[END_REF]. The difference in intensity observed for the peaks corresponding to -OH groups (i.e. bending at 1630 cm -1 and stretching around 2800-3700 cm -1 ) follows the same explanation [START_REF] Mansur | FTIR and UV-vis study of chemically engineered biomaterial surfaces for protein immobilization[END_REF][START_REF] Péré | Quantitative assessment of organic compounds adsorbed on silica gel by FTIR and UV-VIS spectroscopies: the contribution of diffuse reflectance spectroscopy[END_REF][START_REF] Antony | Highly dispersed Cu(II), Co(II) and Ni(II) catalysts covalently immobilized on imine-modified silica for cyclohexane oxidation with hydrogen peroxide[END_REF][START_REF] Capeletti | Infrared and Raman spectroscopic characterization of some organic substituted hybrid silicas[END_REF][START_REF] Célino | Qualitative and quantitative assessment of water sorption in natural fibres using ATR-FTIR spectroscopy[END_REF]. This difference is also explained by the desorption of residual water during the calcination process. Peaks corresponding to the surfactant are observed for the untreated sample, i.e. C-H bond (i.e. bending at 1345 cm -1 and 1450 cm -1 ), -CH 2 groups (i.e.

bending at 1345 cm -1 and 1450 cm -1 and stretching around 2880 cm -1 and at 2935 cm -1 ), -CH 3 groups (i.e. bending at 1375 cm -1 , and stretching around 2880 cm -1 and at 2970 cm -1 ) [START_REF] Capeletti | Infrared and Raman spectroscopic characterization of some organic substituted hybrid silicas[END_REF][START_REF] Adumeau | Impact of surface grafting density of PEG macromolecules on dually fluorescent silica nanoparticles used for the in vivo imaging of subcutaneous tumors[END_REF][START_REF] Bharathi | Direct Synthesis and Characterization of Gold and Other Noble Metal Nanodispersions in Sol -Gel-Derived Organically Modified Silicates[END_REF].

The intensity of these peaks greatly decreases after the first acidic treatment (i.e. 4.9-fold decrease in the area under the curve compared to the untreated sample). These peaks are nevertheless still detected, suggesting the presence of residual surfactant in the pores. In order to reduce the quantity of residual surfactant, a second acidic treatment is performed. This process further decreases the intensity of the peaks (i.e. 10.4-fold decrease in the area under the curve compared to the untreated sample) but does not completely remove the surfactant. On the contrary, after calcination, these peaks totally disappear, indicating that surfactant is not present in the pores anymore.

The efficiency of the surfactant removal technique is also determined by TGA (Figure 2 (d)). All samples exhibit similar TGA curves with three consecutive weight losses. The weight loss between 30 and 150 °C (i.e. 3-8 wt.%) is assigned to the removal of adsorbed water [START_REF] Laird | Single-template periodic mesoporous organosilica with organized bimodal mesoporosity[END_REF].

Between 150 and 320-350 °C, a second weight loss (i.e. 23 wt.% for the untreated sample, 4

wt.% after one acidic treatment, 2 wt.% after two acidic treatment, and < 1 wt.% after calcination) is due to the degradation of residual surfactant in the pores. These results confirm the ones obtained from the FTIR analysis. Indeed, the surfactant content greatly decreases after the acidic treatment (i.e. 6.2 and 10.5-fold decrease after one and two acidic treatments respectively, which is similar to the decrease in area under the curve observed in the FTIR analysis) but residual surfactant remains inside the pores. On the opposite, no weight loss is detected between 150 and 350 °C for the sample after calcination, indicating the absence of surfactant. The last weight loss (i.e. between 320-350 and 900 °C) corresponds to the condensation of adjacent silanols into siloxane bonds and therefore to the presence of -OH groups. As already mentioned, the lower proportion of -OH groups in the calcined sample (i.e. 2 wt.%) compared to the untreated one and the ones treated with acid (i.e. 5 wt.%), can be explained by the condensation of adjacent silanols into siloxane bonds during the calcination at 550 °C [START_REF] Warring | Sur fi cial Siloxane-to-Silanol Interconversion during Room-Temperature Hydration/Dehydration of Amorphous Silica Films Observed by ATR-IR and TIR-Raman Spectroscopy[END_REF][START_REF] Etienne | Analytical investigation of the chemical reactivity and stability of aminopropyl-grafted silica in aqueous medium[END_REF]. These values correspond to a surface concentration of -OH groups of 5.2, 3.5, 5.6, and 6.4 -OH groups/nm² for the sample before treatment, TEOS-1, TEOS-2, TEOS-3, and TEOS-4 respectively, which is in agreement with those obtained by Zhuravlev et al. [START_REF] Zhuravlev | Concentration of Hydroxyl Groups on the Surface of Amorphous Silicas[END_REF].

Figure 2 -(a-c) FTIR spectra: (a) zone 400-1300 cm -1 , (b) zone 1250-1750 cm -1 , (c) zone 2600-3800 cm -1 ; and (d) TGA curves of the TEOS samples:  before treatment,  after calcination (TEOS-1),  after the first acidic treatment (TEOS-2),  after the second acidic treatment (TEOS-3).

The FTIR absorption spectrum of the BTEB sample is displayed in Figure 3 (a-c). The FTIR spectrum exhibits characteristic peaks of silica. Interestingly, these values are shifted towards lower wavenumbers compared to the TEOS samples. The peaks corresponding to the bending of Si-O-Si groups are observed below 400 cm -1 [START_REF] Mansur | FTIR and UV-vis study of chemically engineered biomaterial surfaces for protein immobilization[END_REF][START_REF] Péré | Quantitative assessment of organic compounds adsorbed on silica gel by FTIR and UV-VIS spectroscopies: the contribution of diffuse reflectance spectroscopy[END_REF][START_REF] Antony | Highly dispersed Cu(II), Co(II) and Ni(II) catalysts covalently immobilized on imine-modified silica for cyclohexane oxidation with hydrogen peroxide[END_REF]. The stretching of Si-O-Si groups is detected at 775 cm -1 (i.e. symmetric stretching), at 1040-1045 cm -1 (i.e. transversal optic mode of asymmetric stretching), and in the 1090-1200 cm -1 (i.e. longitudinal optic mode of asymmetric stretching) [START_REF] Péré | Quantitative assessment of organic compounds adsorbed on silica gel by FTIR and UV-VIS spectroscopies: the contribution of diffuse reflectance spectroscopy[END_REF][START_REF] Antony | Highly dispersed Cu(II), Co(II) and Ni(II) catalysts covalently immobilized on imine-modified silica for cyclohexane oxidation with hydrogen peroxide[END_REF][START_REF] Capeletti | Infrared and Raman spectroscopic characterization of some organic substituted hybrid silicas[END_REF][START_REF] Osswald | FTIR spectroscopic study on liquid silica solutions and nanoscale particle size determination[END_REF]. Characteristics peaks of silanol groups are found at 520 cm -1 (i.e. rocking mode of Si-OH groups) and around 915 cm -1 (i.e. asymmetric stretching of Si-OH groups) [START_REF] Mansur | FTIR and UV-vis study of chemically engineered biomaterial surfaces for protein immobilization[END_REF][START_REF] Antony | Highly dispersed Cu(II), Co(II) and Ni(II) catalysts covalently immobilized on imine-modified silica for cyclohexane oxidation with hydrogen peroxide[END_REF][START_REF] Capeletti | Infrared and Raman spectroscopic characterization of some organic substituted hybrid silicas[END_REF][START_REF] Osswald | FTIR spectroscopic study on liquid silica solutions and nanoscale particle size determination[END_REF]. The shift towards lower wavenumbers is related to the deformation of the network to incorporate the organic groups within the silica matrix (i.e. greater Si-O-Si angles and Si-O bond lengths) [START_REF] Capeletti | Infrared and Raman spectroscopic characterization of some organic substituted hybrid silicas[END_REF]. The phenyl moieties are detected through the -CH groups (i.e. bending around 630, 645, 808, 860, 1152, 1300, and 1380 cm -1 and stretching at 3060 cm -1 ), the aromatic ring (i.e. torsion around 630-645 and 1020 cm -1 ), and -C=Cgroups (i.e. stretching at 1455 cm -1 ) [START_REF] Capeletti | Infrared and Raman spectroscopic characterization of some organic substituted hybrid silicas[END_REF][START_REF] Adumeau | Impact of surface grafting density of PEG macromolecules on dually fluorescent silica nanoparticles used for the in vivo imaging of subcutaneous tumors[END_REF][START_REF] Bharathi | Direct Synthesis and Characterization of Gold and Other Noble Metal Nanodispersions in Sol -Gel-Derived Organically Modified Silicates[END_REF][START_REF] Mohan | FTIR and Raman studies on benzimidazole[END_REF][START_REF] Mary | Quantum mechanical and spectroscopic (FT-IR , FT-Raman , 1H NMR and UV) investigations of 2-(phenoxymethyl) benzimidazole[END_REF][START_REF] Coates | Interpretation of Infrared Spectra , A Practical Approach[END_REF]. The peak at 1205 cm -1 can be attributed to the Si-C bond [START_REF] Capeletti | Infrared and Raman spectroscopic characterization of some organic substituted hybrid silicas[END_REF]. -OH groups are also found (i.e. bending around 1635 cm -1 and stretching around 2700-3700 cm -1 ) [START_REF] Mansur | FTIR and UV-vis study of chemically engineered biomaterial surfaces for protein immobilization[END_REF][START_REF] Péré | Quantitative assessment of organic compounds adsorbed on silica gel by FTIR and UV-VIS spectroscopies: the contribution of diffuse reflectance spectroscopy[END_REF][START_REF] Antony | Highly dispersed Cu(II), Co(II) and Ni(II) catalysts covalently immobilized on imine-modified silica for cyclohexane oxidation with hydrogen peroxide[END_REF][START_REF] Capeletti | Infrared and Raman spectroscopic characterization of some organic substituted hybrid silicas[END_REF][START_REF] Célino | Qualitative and quantitative assessment of water sorption in natural fibres using ATR-FTIR spectroscopy[END_REF]. As for the TEOS samples, the TGA curve of the BTEB sample present a weight loss between 30 and 150 °C (i.e. 8 wt.%) attributed to the removal of water (Figure 3 (d)) [START_REF] Laird | Single-template periodic mesoporous organosilica with organized bimodal mesoporosity[END_REF]. Between 150 and 350 °C, a second weight loss (i.e. 7 wt.%) is observed probably due to the degradation of residual surfactant or unreacted silica precursor (ethoxy groups) in the pores, as it occurs in the same temperature range as for the TEOS samples. This observation explains the presence of peaks corresponding to -CH 2 (i.e. bending at 1240, 1347, and 1454 cm -1 and stretching at 2868 and 2930 cm -1 ) and -CH 3 groups (i.e. bending at 1347 cm -1 and stretching at 2868 and 2977 cm -1 ) in the FTIR spectrum of the BTEB sample. The third weight loss (i.e. between 350 and 500 °C, 4

wt.%) corresponds to the condensation of adjacent silanols into siloxane bonds and therefore to the presence of -OH groups [START_REF] Trens | Evolution of Surface Composition , Porosity , and Surface Area of Glass Fibers in a Moist Atmosphere[END_REF]. This temperature range also corresponds to the degradation of hydrocarbons [START_REF] Keene | Calcination of the MCM-41 mesophase: mechanism of surfactant thermal degradation and evolution of the porosity[END_REF][START_REF] Rose | Thermal oxidative degradation of an epoxy resin[END_REF][START_REF] Temtsin | Aromatic PMOs: tolyl, xylyl and dimethoxyphenyl groups integrated within the channel walls of hexagonal mesoporous silicas[END_REF]. It can therefore be assumed that the degradation of the structure is also initiated at these temperatures through the oxidation of the phenylene groups present in the sample walls. Above 500 °C, the degraded organic species decompose, leading to the last weight loss (i.e. 31 wt.%). The measured proportion of phenylene groups in the materials is obtained by disregarding the weight losses corresponding to the water and the surfactant, i.e. by considering that 100 wt.% of the pure materials is present at 350 °C. Based on this assumption, the proportion of phenylene groups is equivalent to 42 wt.%, which is close to the theoretical calculated value (i.e. 45.8 wt.%).

In the present application, the presence of residual surfactant would not be a concern. Indeed, Poloxamers are accepted by the US Food and Drug Administration [START_REF] Russo | Poloxamer Hydrogels for Biomedical Applications[END_REF][START_REF] Singla | Temperature-Dependent Solubilization of the Spectroscopic , Heat Transfer Method , Small-Angle Neutron Scattering , Dynamic Light Scattering , and in Vitro Release Study[END_REF]. Due to their biocompatibility, Pluronic P-123 has been notably used as a drug delivery vehicle (e.g. calcein, curcumin) [START_REF] Bhattacharjee | Microstructure, drug binding and cytotoxicity of Pluronic P123-aerosol OT mixed micelles[END_REF][START_REF] Duan | Novel in situ gel systems based on P123 / TPGS mixed micelles and gellan gum for ophthalmic delivery of curcumin[END_REF][START_REF] Zhao | Recent progress in drug delivery of pluronic P123 : pharmaceutical perspectives[END_REF].

Textural properties

The nitrogen adsorption-desorption isotherms are depicted in Figure 4, while the textural properties of the samples are given in Table 3. For all samples, a Type IV isotherms according to the IUPAC classification is present, indicating the presence of micro-and mesopores [START_REF] Thommes | Physisorption of gases, with special reference to the evaluation of surface area and pore size distribution (IUPAC Technical Report)[END_REF]. Type IV isotherms are characterized by the presence of a hysteresis resulting from capillary condensation occurring inside the mesopores of the material. Two types of hysteresis loops are observed. On the one hand, the Type H1 hysteresis found for the TEOS-1, TEOS-2, and BTEB samples is characterized by hysteresis loop with steep and parallel adsorption/desorption branches, indicating the presence of a narrow range of uniform mesopore sizes. A shift of the hysteresis towards lower pressure is observed from the TEOS-1 sample to the BTEB one, indicating a lower pore size for the BTEB sample (i.e. from 9.8 nm for the TEOS-1 and TEOS-2 samples to 5.1 nm for the BTEB one). When comparing the TEOS-1 and TEOS-2 samples, it is observed that the presence of residual surfactant inside the silica pores does not significantly influence the textural properties of the sample, as both samples exhibit similar isotherms and pore size distributions. The slight difference in specific surface area value (i.e. 585 m²/g for the TEOS-1 sample vs 505 m/g for the TEOS-2 one) between these two samples could be explained by the fact that the residual surfactant trapped inside the pores. Due to the slight difference in textural properties between the sample after the acidic treatment and the calcination, it was decided to not further explore the TEOS-3 sample. On the other hand, the shape of the hysteresis of the TEOS-4 sample (i.e. Type H2) points out the presence of disorganized mesopores, suggesting the presence of porous but unstructured silica.

This observation shows that the time of TIPB addition greatly affects the textural properties, with the presence of structured silica for the TEOS-1 sample (i.e. addition of TIPB during TEOS addition) and unstructured silica for the TEOS-4 sample (i.e. addition of TIPB during P123 and NH 4 F dissolution). This observation is confirmed by the pore size distribution with a narrow distribution for the TEOS-1 sample (i.e. centered at 9.8 nm) compared to a wider pore size distribution for the TEOS-4 sample, going from 3 nm to 12 nm (Figure 4 (b)).. These differences in structure are probably due to the silica network formation mechanism.

Without the presence of a swelling agent as TIPB at the beginning of the synthesis, spherical micelles and hydrolyzed TEOS are allowed to interact and their interaction progressively form cylindrical micelles (at the given experimental conditions) where the core is more hydrophobic and the cylindrical surface is more hydrophilic [START_REF] Manet | Kinetics of the Formation of 2D-Hexagonal Silica Nanostructured Materials by Nonionic Block Copolymer Templating in Solution[END_REF][START_REF] Sundblom | On the Formation Mechanism of Pluronic-Templated Mesostructured Silica[END_REF]. Hydrolyzed TEOS can therefore condense via sol-gel reactions into a silica network around this hydrophilic cylindrical surface. These micellar aggregates then grow via the incorporation of more silica precursor into the network. As silica is incorporated, micelles associate and form particles, eventually leading to a 2D-hexagonal structure. When TIPB and TEOS are added simultaneously and gradually (dropwise addition), TEOS is rapidly hydrolyzed (acid catalysis and high content in water), which allows its direct association with the spherical micelles. The TEOS precursor then plays a stabilization role of the micelle while the TIPB is added in increasing quantity. The expansion of the micelle is concomitant to the stabilization with the hydrolyzed TEOS thus preserving the 2Dhexagonal structure but with larger pores. By contrast, when added during the dissolution of P123 step, a large quantity of TIPB is allowed to interact with the hydrophobic part of P123 (without stabilization effect of the TEOS), and may prevent the formation of the spherical initiating micelles. In this case, the later addition of TEOS does not trigger the organization of the mesophase, which leads to the condensation of TEOS without soft templating. A disordered silica material is formed. Note: S BET : specific surface area determined by the BET method; V P : specific liquid volume adsorbed at the saturation pressure of nitrogen; V micro : specific microporous volume; V meso : specific mesoporous volume; V meso : specific mesoporous surface; d BJH : mean pore size determined by the BJH theory.

The SAXS patterns of the structured samples (i.e. TEOS-1, TEOS-2, and BTEB samples) are illustrated in Figure 5. The pattern of the TEOS-1 and TEOS-2 samples exhibits five pseudo-Bragg peaks in the low q region with q 0 equal to 4.9 x 10 -2 and 4.6 x 10 -2 Å -1 respectively. The relative position of the peaks corresponds to the (100), ( 110), ( 200), (210), and (300) reflections from a 2D-hexagonal mesostructure (P6 3 /m symmetry) with a lattice parameter a SAXS equal to 14.8 nm for the TEOS-1 sample and to 15.5 nm for the TEOS-2 one [START_REF] Cao | Synthesis of Ultra-Large-Pore SBA-15 Silica with Two-Dimensional Hexagonal Structure Using Triisopropylbenzene As Micelle Expander[END_REF][START_REF] Laird | Single-template periodic mesoporous organosilica with organized bimodal mesoporosity[END_REF]. The fact that the (210) and (300) reflections can be seen shows that the silica is highly organized. By contrast, the SAXS pattern of the BTEB sample exhibits two sets of peaks. The first set (q 0 equal to 7.2 x 10 -2 Å -1 , indicated with the symbol #) corresponds to the (100), (110), and (200) reflections from a 2D-hexagonal structure with a lattice parameter a SAXS = 10.1 nm. The second set (q 0 equal to 8.6

x 10 -2 Å -1 , indicated with a symbol *) corresponds to the (100), (110), and (200) reflections from a 2D-hexagonal structure with a smaller lattice parameter, i.e. a SAXS = 8.6 nm.

The TEM analysis corroborates the nitrogen adsorption-desorption and SAXS measurements (Figure 6, Table 4). The TEOS-1 and TEOS-2 samples exhibit an organized porosity with hexagonal symmetry, as shown by the honeycomb structures observed. The corresponding FFT data (inserts in Figure 6, larger representation is available in Supplementary Materials, Figure S2) are consistent with hexagonal symmetry, as characteristic hexagonal features are observed. The apparent wall thickness, calculated from the lattice parameter a TEM and the pore diameter determined via nitrogen adsorption, d BJH , is similar for the two samples, i.e. close to 3.6 nm. A similar honeycomb structure is observed for the BTEB sample, indicating an organized porosity with hexagonal symmetry. Yet, as shown by the superposition of two series of characteristic hexagonal features in the FTT data, it appears that two hexagonal structures with different lattice parameters are present. Taking into account that the BTEB sample only exhibits one pore size, these two structures correspond to two different wall thicknesses. Unfortunately, only one lattice parameter could be found. It is hypothesized that, due to the small difference between the lattice parameter values, the two structures could not be differentiated with the image treatment and that the value obtained is a mean of the two values. In contrast to these structured silica samples, the TEOS-4 sample does not present a honeycomb feature but is rather composed of spherical elementary particles.

Protein encapsulation

The amount of encapsulated STI per unit mass of silica and per unit of specific surface area of silica are presented in Figure 7. The protein is encapsulated on the TEOS-1 and TEOS-4 samples, in order to study the effect of the silica structure on the encapsulation of the protein. It is important to keep in mind that these samples were synthesized using the same synthesis protocol (except for the time of addition of TIPB) and that great care was taken to ensure identical experimental parameters, i.e. pH, solvent, temperature and stirring (cf. section 2.3.1). The BTEB sample is also considered for the protein encapsulation in order to study the influence of the presence of phenylene groups within the sample walls.

The amount of encapsulated STI greatly varies among samples. A larger protein loading is observed for the unstructured TEOS-4 sample (38 mg/g) when compared to the structured ones (19 mg/g for the TEOS-1 sample and 21 mg/g for the BTEB one), regardless of the silica precursor nature. This can be explained by a blockage of the pores by the proteins [START_REF] Zhou | Mesoporous silica nanoparticles for drug and gene delivery[END_REF][START_REF] Serra | A comparative study of periodic mesoporous organosilica and different hydrophobic mesoporous silicas for lipase immobilization[END_REF].

Indeed, even if the pore were to be larger than the STI average radius (about 4 nm), the adsorption of two protein molecules on the same pore section would hinder the pore, preventing further protein insertion inside this pore. In the case of ordered silica materials, the blockage of the two extremities of the pore seals the entire pore off, greatly decreasing the surface area accessible for protein adsorption. On the opposite, unstructured silica (i.e. TEOS-4 sample) is composed of an interconnected network of pores. Therefore, if a pore is blocked on one side, the proteins can diffuse through other paths. The amount of adsorbed STI in "per specific surface area" of each sample (Figure 7(b)) follow the same rationale. The values for the TEOS-4 sample are similar to those previously observed for calcined unstructured silica [START_REF] Tilkin | Protein encapsulation in functionalized sol -gel silica : influence of organosilanes and main silica precursors[END_REF]. It is important to note that these results represent the total amount of protein, i.e. inside and at the external surface of the samples. Considering the results of this section, it seems that the proteins present at the surface of the silica account for 20 to 35 % of the total amount. This means that the quantity of encapsulated proteins is equal to 15 mg/g for the TEOS-1 sample and to 26 mg/g for the TEOS-4 one.

Release kinetics profiles

Assuming that the burst is probably due to STI adsorbed at the surface, we have decided to remove the part corresponding to the premature release in order to focus on the release of the protein encapsulated inside the silica pores. We therefore express the rest of the profile as the proportion of the remaining STI after 24 h that is released in the medium (Figure 8(c)). Beyond this period, the STI release slows down for the TEOS-4 sample. The release profile seems to achieve a plateau, as observed in previous studies [START_REF] Tilkin | Protein encapsulation in functionalized sol-gel silica : Effect of the encapsulation method on the release kinetics and the activity[END_REF][START_REF] Tilkin | Protein encapsulation in functionalized sol -gel silica : influence of organosilanes and main silica precursors[END_REF]. The STI retention might be linked to the interactions between the protein and silanol groups present at the surface of the pores, preventing the total release of the protein. By contrast, a sustained release of STI is observed up to day 42 for TEOS-1, as the release profile does not seem to reach a plateau within the studied period. These results indicate that structured silica can produce a sustained release on a longer period than porous unstructured silica. This difference can be explained by the pore interconnectivity [START_REF] Serra | A comparative study of periodic mesoporous organosilica and different hydrophobic mesoporous silicas for lipase immobilization[END_REF][START_REF] Vallet-Regí | Mesoporous Silica Nanoparticles for Drug Delivery : Current Insights[END_REF]. In the case of structured silica (i.e. TEOS-1 sample), the proteins can only diffuse through the two extremities of a given pore, since the STI size does not allow it to diffuse through micropore channels that connect the mesoporous cylinders together. Therefore, proteins that are adsorbed at the centre of a pore cannot easily diffuse outside as long as there are proteins in their path to the exit. On the opposite, the unstructured sample presents an interconnected network of mesopores. In this case, the proteins can bypass possible pore blockage, as they would have more opportunities of escaping the silica matrix and be released.

In contrast with the TEOS samples, the BTEB sample does not present a significant release of STI over the 30 days period. These observations corroborates the high affinity of the STI for the phenylene groups, as previously shown [START_REF] Tilkin | Protein encapsulation in functionalized sol -gel silica : influence of organosilanes and main silica precursors[END_REF]. It is important to notice that phenylene groups represented a high proportion of the materials (i.e. 22 wt.% in [START_REF] Tilkin | Protein encapsulation in functionalized sol -gel silica : influence of organosilanes and main silica precursors[END_REF] and 42 wt.% in this present study). It is expected that an adequate balance of hydrophobic/hydrophilic groups could enable a tailored release kinetic of the STI protein to be obtained by controlling the hydrophobicity of the material with the BTEB content. This can be envisioned by inserting polar organic groups (such as amino groups) in the pore walls which will surely allow more hydrophilic characteristics and favour the release of STI.

CONCLUSIONS

In conclusion, SBA-15 type silica samples were first synthesized using TEOS as silica precursor in the presence of a swelling agent to increase the pore size. Two different surfactant removal treatments were investigated, i.e. calcination and acidic treatment. The latter treatment did not completely remove the surfactant, while no trace of residual surfactant was detected in the calcined samples. Yet, the samples produced with these two techniques exhibited similar textural morphological properties as well as 2D-hexagonal honeycomb mesostructures. The reagent addition sequence was also investigated, specifically by playing on the time of addition of a swelling agent (TIPB). The textural analysis showed the presence of organization in the sample for which the swelling agent was added together with the TEOS. On the opposite, when the swelling agent was added during the surfactant dissolution, porous unstructured silica was produced.

Based on the high affinity of STI for hydrophobic surfaces, SMS were also produced using BTEB to incorporate phenylene groups inside the wall matrix. In contrast with the TEOS samples, the BTEB sample exhibited two hexagonal structures with different wall thicknesses, as

shown by the TEM and SAXS analyses.

The influence of the silica structure (i.e. ordered vs disordered) and composition (i.e. TEOS vs BTEB) on STI encapsulation and release was finally studied. The higher protein loading observed for the unstructured sample (compared to the structured materials regardless of the nature of the silica precursor) was explained by a difference in the pore interconnectivity among the samples. Regarding the release profiles, for the TEOS samples, a fast release of STI was observed during the first 24 h. Beyond this period, the STI release slowed down for the unstructured sample and seemed to reach a plateau, while a sustained release of STI was observed up to day 28 for the structured sample. These results were also explained by a difference in pore interconnectivity thus highlighting the capacity of the ordered silica to release the protein on a more gradual manner. On the opposite, the BTEB sample did not show any significant release of STI over the 30 days period, because of a higher affinity of the protein towards phenylene groups. As perspective, more efforts should be devoted to better adjust the hydrophobicity of the silica surface, in order to optimally reduce the protein binding and enhance its release rate.

ACKNOWLEDGMENTS

Figure 1 -

 1 Figure 1 -Chemical structure of BTEB.

  standard. Background scattering, based on measuring an empty DSC pan, was subtracted taking into account the individual transmission of each sample. The lattice parameter (a SAXS , Å) was determined as an average of unit-cell parameters defined for the (100), (110) and (200) reflections according to Equations 3, 4, and 5, related to a 2D-hexagonal honeycomb structure

Figure 3 -

 3 Figure 3 -(a-c) FTIR spectrum: (a) zone 400-1200 cm -1 , (b) zone 1150-1750 cm -1 , (c) zone 2600-3800 cm -1 and (d) TGA curve of the BTEB sample. Ar = Aromatic ring.

Figure 4 -

 4 Figure 4 -Nitrogen adsorption-desorption analysis of the TEOS and BTEB samples. (a) Adsorption-desorption isotherms and (b) pore size distributions:  TEOS-1,  TEOS-2,  TEOS-4,  BTEB.

Figure 5 -

 5 Figure 5 -SAXS analysis of the  TEOS-1,  TEOS-2,  BTEB samples. For the BTEB sample, the first set of peaks is indicated with the symbol # and the second one with the symbol *.

Figure 6 -

 6 Figure 6 -TEM images of the (a-b) TEOS-1, (c-d) TEOS-2, (e-f) TEOS-4, and (g-h) BTEB samples. The inserts in (b-d-f-h) are the corresponding FFT.

Figure 7 -

 7 Figure 7 -STI loading expressed as (a) m STI /m Sample and (b) m STI /S BET;Sample in the TEOS and BTEB samples:  TEOS-1,  TEOS-4,  BTEB.

Figure 8

 8 Figure8illustrates the release kinetic profiles. For the sake of clarity, Figure8was divided into

Figure 8 -

 8 Figure 8 -Release kinetics profile of STI from the TEOS and BTEB materials: (a) Release kinetics profile over 45 days (b) Initial release profile (0 to 24 h) (c) Profile of the STI released after the first day (STI release up to 24 h is subtracted):  TEOS-1,  TEOS-4,  BTEB.

Table 1 -

 1 Properties of STI and BMP-2

	Properties	STI	BMP-2
	Molecular weight (g/mol)

  (ΔA 253 ) of 0.001/min with BAEE as substrate at pH = 7.6 and at 25 °C.

	Tetraethyl orthosilicate (TEOS, Si(OC 2 H 5 ) 4 ) potassium phosphate monobasic (KH 2 PO 4 ), sodium
	chloride (NaCl), potassium chloride (KCl), 1,2-dibromoethane, pentane (≥ 95%), 1,4-
	dibromobenzene (98 %), deuterochloroform (CDCl 3 ), tetrahydrofuran (THF), and Pluronic P123
	were purchased from Sigma-Aldrich; hydrochloric acid (HCl (37 %)), absolute ethanol, and
	sodium hydroxide (NaOH) were obtained from Merck; ammonium fluoride (NH 4 F), sodium
	phosphate dibasic (Na 2 HPO 4 ), and sodium phosphate monobasic (NaH 2 PO 4 ) were provided by
	Acros Organics; Soybean Trypsin Inhibitor STI (≥ 7000 BAEE U/mg) was purchased from Carl
	Roth; 1,3,5-triisopropylbenzene (TIPB, 95 %) was obtained from Alfa Aesar; Maleic acid
	(C 4 H 4 O 4 ) was purchased from Sigma. Unless indicated otherwise, all these chemicals were ≥ 99
	% pure and were used without further purification. Phosphate Buffer Saline (PBS) solution was
	prepared using 1.4 mM KH 2 PO 4 , 10 mM Na 2 HPO 4 , 137 mM NaCl and 2.7 mM KCl and was
	adjusted at pH = 7.4 using 0.1 M HCl or 0.1 M NaOH.

Table 3 -

 3 Textural properties of the samples.

	Sample

S BET (m²/g) ± 5 V P (cm³/g) ± 0.1 V micro (cm³/g) ± 0.01 V meso (cm³/g) ± 0.01 d BJH (nm)

	TEOS-1	585	1.3	0.60	0.70	9.8
	TEOS-2	505	1.3	0.66	0.64	9.8
	TEOS-4	515	1.1	0.25	0.85	6.8
	BTEB	700	0.9	0.53	0.37	5.1

Table 4 -

 4 Textural properties of the organized samples (TEM and SAXS analysis).

  TEM and t TEM : lattice parameter (pore size + wall size) and wall size determined by TEM; a SAXS and t SAXS : lattice parameter (pore size + wall size) and wall size determined by SAXS.

			TEM analysis	SAXS analysis
		Sample	a TEM	t TEM	a SAXS	t SAXS
			(nm)	(nm)	(nm)	(nm)
	1.E+9	(100) TEOS-1	13.4 ± 0.5	4.1	14.8	5.5
	1.E+8	(100) TEOS-2	(110) 13.5 ± 1.1 (200) (210)	4.2	15.5	6.2
	1.E+5 1.E+6 1.E+7 Note: a Intensity (a.u.)	BTEB	(110) # (110)* (200) # (300) (200) (300) (100)* (110) 9.1 ± 0.4 4.3 (210) (100) #	8.6-10.1 3.8-5.3
	1.E+4		(200)*
	1.E+3
	1.E-2		1.E-1		1.E+0
			q (Å -1 )
	(a)	(c)		(e)		(g)
		100 nm	100 nm		100 nm	100 nm
	(b)	(d)		(f)		(h)
		50 nm	50 nm		50 nm	50 nm

Funding: This work was supported by the Belgian Fund for Scientific Research (F.R.S.-FNRS) under a Fund for Research Training in Industry and Agriculture (FRIA) grant, the "Ministère wallon de la Recherche et de l'Innovation", and the Wallonia region.

Data availability

The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.

The authors thank Dr. Ir. Cedric J. Gommes for his help to process the SAXS data and for fruitful and interesting discussions.

The authors thank Camille Tilkin for her corrections.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest concerning this work.