WO2022013250A1

WO2022013250A1 - Process for synthesizing nanoscale and sub-micron-sized porous silica particles by soft chemistry

Info

Publication number: WO2022013250A1
Application number: PCT/EP2021/069520
Authority: WO
Inventors: Karine Fabio; Marlene SAUVAT; Julien SALDO; Christophe Di Giorgio; Franck CHUZEL
Original assignee: Lifescientis; Universite Cote D'azur; Centre National De La Recherche Scientifique
Priority date: 2020-07-16
Filing date: 2021-07-13
Publication date: 2022-01-20
Also published as: FR3112539A1; FR3112539B1

Abstract

The invention provides a process for synthesizing nanoscale or sub-micron-sized porous silica particles, comprising the following steps: a) preparing silica nuclei by hydrolysing a silica precursor in water in an acid-catalytic medium, the amount of acid being selected preferably such that it is not more than 0.1 equivalent of acid, then b) forming a matrix phase by mixing at least one condensation agent, and at least one bile acid, or salt thereof, then adding at least one monovalent, divalent or trivalent phosphate anion to the mixture comprising the condensation agent and bile salt, c) condensing the silica nuclei obtained in step a) in a basic medium, by mixing, with stirring, the silica nuclei obtained in step a) with the matrix phase obtained in step b) at an alkaline pH of not more than 10. The present invention relates to the field of synthesis by soft chemistry and more particularly to the preparation of nanoscale or sub-micron-sized silica particles.

Description

Process for the synthesis of nanometric and submicron porous silica particles by soft chemistry

TECHNICAL AREA

The present invention relates to the field of synthesis by soft chemistry and more particularly to the preparation of nanometric or submicron silica particles.

STATE OF THE ART

Silica and silica-based porous materials have many advantages in various industrial, technological and medical application fields. Examples of non-limited applications include semiconductors, catalysis, chromatographic separation techniques, dietary supplements, drug delivery, human or veterinary cosmetics. This wide variety of applications is partly explained by the development of tools which make it possible to obtain silicas of very varied size, morphology and properties (hexagonal, fibers, films, functionalized materials, etc.) directly adapted to the applications. wanted.

Porous materials based on amorphous silica in nanometric or submicron form are tools particularly suited to the delivery of active ingredients in humans or animals because they are recognized as non-toxic, biocompatible and biodegradable. Porous silicas, in particular of the mesoporous MCM41 or SBA15 type, have continued to gain interest in this field. Their syntheses are based on a known and very widely described principle based on the cooperative assembly of surfactant micelles allowing the condensation of silica around these organized structures. Conventional Stôber-type sol-gel chemistry conditions for the hydrolysis of a silica precursor and the condensation of silica take place at very acidic or very alkaline pH, in the presence of an organic solvent, at high temperatures in the presence of surfactants such as non-natural quaternary ammonium (CTAB), which must be totally eliminated to release the porosity and limit the risks of toxicity.

So-called soft chemistry is increasingly sought after to develop synthetic processes that are more ecological and that integrate more harmoniously into natural processes.

Examples of porous silica preparation using natural surfactants exist. Patent WO 2017/182245, for example, describes the use of natural structuring agents from the saponin family as a replacement for petroleum-based detergents.

We know from the document - XP055451175 - WENLANG LIANG et Al. "Morphology and Shape Control of Porous Silica Nanostructures with Dual Templating Approaches" Journal of Nanoscience and nanotechnology. Flight. 14 no.6 June 1, 2014 - pages 4424-4430, the synthesis of porous silica nanostructures in a two-phase system comprising hexane. This type of synthesis does not meet the need for a process that is more respectful of the environment.

However, there is a need to propose a synthesis of porous silica particles according to soft chemistry methods.

An object of the present invention is therefore to provide a process for the synthesis of silica particles of controlled nanometric or submicron size which is more ecological and less energy-consuming than the conventional processes of the state of the art.

The other objects, features and advantages of the present invention will become apparent from a review of the following description and the accompanying drawings. It is understood that other benefits may be incorporated.

ABSTRACT

To achieve this objective, according to one embodiment, provision is made

A process for the synthesis of nanometric or submicron porous silica particles comprising the following steps: a) preparation of silica nuclei by hydrolysis of a silica precursor in water in an acid-catalyzed medium, preferably the quantity of acid is chosen of so as to be less than or equal to 0.1 equivalent of acid, more preferably between 0.1 and 0.001, more preferably equal to 0.001 eq of acid, preferably the acid is chosen from a weak carboxylic acid or a strong acid for example formic acid or acetic acid or hydrochloric acid, then b) formation of a matrix phase by mixing at least one condensing agent advantageously chosen from a basic polycationic polycationic polymer that is branched or linear, and of at least one bile acid, or its salt, then addition of at least one monovalent, divalent, trivalent anion advantageously chosen from a phosphate salt, a citrate salt, a tartrate salt, a sulphate salt, or a salt of nitrate to the mixture comprising, preferably composed of, the condensing agent and bile acid, then c) condensation in a basic medium of the silica nuclei obtained in step a) by mixing with stirring the silica nuclei obtained in step step a) and pha matrix obtained in step b) at alkaline pH less than or equal to 10, conventionally called moderate or low pH, preferably pH less than or equal to 9, preferably pH less than or equal to 8.

The process according to the invention allows the synthesis of porous silica particles under mild chemical conditions with in particular a synthesis in water and a condensation at a moderate alkaline pH. In addition, a bile acid or its salt is used as a surfactant, and bile acid or its salt is a natural, non-toxic surfactant that can be partially or completely removed by gentle extraction methods.

The particles obtained by the present process advantageously have a controlled and controlled porosity allowing control of the diffusion of an active ingredient. Advantageously, the porous silica has a minimum specific surface of 20 to 30 m ² /g preferably up to 500m ² /g, for example 80m ² /g.

The process according to the invention uses a polycationic polymer as a matrix for the condensation of silica precursors mimicking natural processes and natural detergents, advantageously endogenous such as bile acids or their salts as a pore-forming agent for the newly formed silica.

The present method of synthesis is inexpensive, efficient, gentle and rapid.

The process implemented is biomimetic and respects soft chemistry conditions.

Advantageously, the method does not include any organic solvent. Yields range from good to excellent (min 30%, average 50% up to 90%). Moreover, the bile acids or their salts used in an original way in the process as pore-forming agents are endogenous molecules. They can therefore be considered non-toxic. The method according to the invention makes it possible to obtain directly, in situ, the formation of silica particles, unlike the known prior art which does not result in a porous silica in the form of particles.

According to an advantageous possibility, the bile acids or their salts are used at concentrations lower than their Critical Micellar Concentration (CMC) which makes it possible to obtain porous particles using less surfactant.

BRIEF DESCRIPTION OF FIGURES

The aims, objects, as well as the characteristics and advantages of the invention will emerge better from the detailed description of an embodiment of the latter which is illustrated by the following accompanying drawings in which:

Figures 1A to 1C are representative TEM images of porous silica particles from Example 1 after heat treatment in a dilute ammonia solution min 1% (Figure 1A), in a saturated bicarbonate solution (Figure 1B) and after calcination at 650° C. (FIG. 1C).

Figure 2 represents the experimental isotherm of adsorption and desorption of dinitrogen at 77 K for example 1 (The measurements of the isotherms of adsorption and those of desorption of dinitrogen (N ₂ ) at 77K (196 ° C) have were carried out on a Micromeritics TRISTAR II 3020 type volumetric apparatus).

FIG. 3 represents the representative distribution curve of the zeta potential of the porous silica particles obtained in example 1.

Figures 4A to 4B are representative images by scanning electron microscopy and after calcination (Figure 4A: magnification X50,000) (Figure 4B: magnification X10,000) of Example 2.

Figure 5 shows a representative thermogram of the mass loss obtained in the temperature range from 25 to 800°C with a ramp of 10°C/min for example 2.

Figures 6A to 6B are representative TEM images of porous silica particles obtained in Example 3 before (Figure 6A) and after heat treatment in ammonia (Figure 6B).

Figures 7A to 7B are representative TEM images of porous silica particles obtained in Example 4 before (Figure 7A) and after heat treatment in ammonia (Figure 7B).

Figures 8A-8D are representative TEM images of silica particles obtained in Example 5 with 15 mM (Example 5A - Figure 8A) or 30 mM (Example 5B - Figure 8B) of APTES (3-aminopropyl)triethoxysilane), respectively added after 20 minutes of stirring with 15 mM (Example 5C - figure 8C) or 30 mM (example 5D - figure 8D)

Figure 9 shows a representative TEM image of porous silica particles obtained in Example 6.

Figure 10 shows a representative TEM image of porous silica particles obtained in Example 7.

The drawings are given by way of examples and do not limit the invention. They constitute schematic representations of principle intended to facilitate understanding of the invention and are not necessarily scaled to practical applications.

DETAILED DESCRIPTION

Before starting a detailed review of embodiments of the invention, optional characteristics are set out below which may possibly be used in combination or alternatively:

According to one example, steps a) to c) are carried out at ambient temperature.

According to one example, the bile acid is selected from cholic acid, taurocholic acid, glycocholic acid, ursocholic acid, glycoursocholic acid, hyocholic acid, glycohyocholic acid, chenodeoxycholic acid , glycochenodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, hyodeoxycholic acid, glycohyodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid or a salt thereof or a mixture thereof.

According to one example, the silica precursor is chosen from at least one of TEOS (tetraethyl orthosilicate), TMOS (tetramethyl orthosilicate), MTMOS (methyltrimethoxysilane), MTEOS (methyltriethoxysilane), ETEOS (ethyltriethoxysilane), ETMOS (ethyltrimethoxysilane) , SUITABLE ((3-Aminopropyl)triethoxysilane), sodium orthosilicate or sodium metasilicate or a mixture.

According to one example, in step a) the silica precursor is in an amount less than or equal to 3000 mM.

According to one example, in step c) the silica precursor is in an amount less than or equal to 1000 mM, preferably less than or equal to 500 mM.

According to one example, the condensation agent is chosen from a polycationic basic polycationic polymer that is branched or linear.

According to one example, the condensation agent is chosen from polyamino acids, polyallylamines, polyethylene imines, derivatives of propylene imines

According to one example, the condensation agent is used in an amount less than or equal to 100 g/L, preferably less than or equal to 50 g/L, preferably less than or equal to 20 g/L.

According to one example, the bile acid or its salt is added in an amount less than or equal to 10 times the Critical Micellar Concentration (CMC), preferably between 0.05 and 10 times the CMC, more precisely between 0.1 and 10 times the CMC.

According to one example, the at least one monovalent, divalent or trivalent anion is added in a concentration less than or equal to 200 mM.

According to one example, the at least one monovalent, divalent or trivalent anion is chosen from at least one of a phosphate salt, a tartrate salt and a citrate salt.

According to one example, the porous silica particles are advantageously spherical.

According to one example, the process comprises, after step c), a step d) of separating the particles and optionally a step e) of drying the porous silica particles obtained and optionally a step f) of purifying the isolated particles.

According to one example, the method comprises a step c′) of functionalizing porous silica particles obtained in step c) by adding a silica precursor, step c′) being simultaneous or successive to step c) and advantageously prior to step d) of separation.

According to one example, step d) of separation is carried out by centrifugation or filtration by tangential route.

According to one example, step e) of drying is carried out by atomization.

According to one example, step f) of purification to eliminate organic residues is carried out by washing, chemical extraction or calcination.

The present invention relates to a process for the synthesis of porous silica particles under mild chemical conditions.

Preferably, the method implements a bio-inspired synthesis.

The particles obtained are advantageously biocompatible and/or biodegradable. This represents an undeniable advantage for the targeted applications.

The particles obtained by the process according to the invention are particularly suitable for human or veterinary pharmaceutical, cosmetic, phytosanitary, food-processing or perfumery applications.

The porous silica produced by the present process advantageously has a so-called intermediate, controlled porosity allowing in particular the carrying of active agents. As preferred, the porosity is greater than 50 m ² /g.

Advantageously, the porous silica has a minimum specific surface of 20 to 30 m ² /g, for example 80 m ² /g, preferably up to 500 m ² /g.

According to one embodiment, the method according to the invention makes it possible to orient the porous silica produced in the form of particles of nanometric or submicron size. By nanometric is meant that the particles have their largest dimension less than or equal to 100 nm, more precisely between 0.1 and 100 nm. By submicron is meant that the particles have their largest dimension less than or equal to 1 μm.

The porous silica particles are advantageously produced in a spherical shape. This has an advantage in terms of biocompatibility compared to rod-type forms, or others, which are less well tolerated.

The porous silica particles obtained according to one of the embodiments have a positive surface charge. This represents an advantage in particular in terms of passive targeting of cells and/or tissues/epithelium which have a residual negative surface charge.

The method advantageously comprises 3 steps.

Advantageously, the three steps: step a), step b) and step c) are carried out in an aqueous medium.

According to one embodiment, the method comprises a step a) of preparing the silica nuclei. According to the invention, step a) is a hydrolysis of at least one silica precursor. Step a) is carried out in an aqueous medium and preferably in an acid-catalyzed aqueous medium.

According to one embodiment, the at least one silica precursor is chosen from precursors:

• of the siloxy type corresponding to the formula (R)xSi(0-R1) ₄ -x with R1= (C1-C4) alkyl or hydroxyl group, and

• R= group type alkoxy, hydrogen, linear or branched alkyl or an alkene, which can present a functional group type amine, carboxyl, thiol, hydroxyl, epoxy

• of the silicate type corresponding to the formula (M20) _x (Si02) _y x=1 or 2, y=1 or 2, with M=metal such as Ca, Na for example.

Preferably, the at least one silica precursor is chosen from at least one least among tetraethyl orthosilicate (TEOS), tetramethyl orthosilicate (TMOS), (3-Aminopropyl)triethoxysilane (APTES), sodium metasilicate, sodium orthosilicate, calcium silicate, trimethoxymethylsilane (MTMOS) , triethoxymethylsilane (MTEOS), triethoxysilanetrimethoxysilane, triethoxy(ethyl)silane (ETEOS), risobutyl(trimethoxy)silane, propyl(trimethoxy)silane, sodium orthosilicate or sodium metasilicate.

According to one possibility, the silica precursor is an extract of biogenic silica such as, for example, from residues of rice or diatoms, sodium silicate or a natural source of ortho silicic acid.

Preferably, the amount of silica precursor in step a) is less than or equal to 3000 mM.

Preferably, the amount of silica precursor in step c) is less than or equal to 1000 mM, more preferably less than 500 mM.

According to one embodiment, the amount of acid added is advantageously less than or equal to 0.1 acid equivalent. More specifically, the amount of acid is between 0.001 and 0.1 acid equivalent. As a preferred example, the amount of acid is equal to 0.001 acid equivalent. These low amounts of acid are an advantage. The amounts of acid are given in acid equivalent with respect to the silica precursor.

According to one possibility, the acid is chosen from a weak carboxylic acid or a strong acid. Preferably, the acid is chosen from formic acid, acetic acid or hydrochloric acid.

Advantageously, the pH of the aqueous medium in which the nuclei are prepared is less than or equal to 5, preferably less than or equal to 4, more preferably, the pH is less than or equal to 3. This pH is adjusted using the amount minimum acid required so as to promote mild chemical conditions in the process.

By way of example, the silica nuclei formed have a size of the order of a few nm in diameter and preferably less than 20 nm, preferably less than 10 nm, more preferably less than 5 nm. The small size favors a maximum entropy which allows obtaining a greater number of particles as well as a better homogeneity in terms of size and spherical shape.

According to one embodiment, the method comprises a step b) of forming a matrix phase. The matrix phase is understood as the phase intended to allow the formation of the porous silica. The matrix is formed by mixing at least one condensing agent and at least one bile acid or its salt. Then this mixture is added at least one monovalent, divalent or trivalent anion or a mixture of monovalent, divalent or trivalent anions.

The condensation agent is advantageously a basic polycationic polymer. This represents an advantage compared to the more traditional use of CTAB-type cationic surfactants.

Preferably, the polymer is chosen to have a weight less than or equal to 800 KDa. According to one aspect, said polymer comprises in particular basic polyamino acids such as polyarginines, polylysines and polyhistidines, but also polyallylamines, polyethylene imines (PEI), polypropylene imines. The condensation agent can result from the chemical modification of one of the polymers mentioned above in order to modulate its physico-chemical properties.

According to one aspect, the condensation agent is more precisely a polymer rich in primary, secondary or tertiary amine functions, that is to say comprising a number of amine residues greater than or equal to 4.

In the case of branched or linear polyethylene imines (PEI), they consist of the repetitive unit ethylene imine type (C2H5N)n with a molar mass of 43.04 g/mol. By way of example, a polymer can be diethylenetriamine or any of its higher homologs.

The condensation agent can also be a branched PEI of the following formula: H(NHCH ₂ CH2)nNH ₂ )n with a molecular weight of between 10,000 and 750,000, in particular between 25,000 and 750,000.

According to one aspect, the condensing agent is a polyamine dendrimer of generation greater than 1 containing [-CH ₂ CH ₂ N(CH ₂ CH ₂ CH ₂ NH ₂ ) ₂ ] _{2 type units} , for example DAB -Am-4, Polypropyleneimine tetramine dendrimer, generation 1.

Preferably, the condensation agent is added during step b) to reach a final mass concentration in the matrix of less than or equal to 100 g/L, preferably less than or equal to 50 g/L, preferably less than or equal to 20 g/L. The process according to the invention makes it possible to use a small quantity of condensing agent and therefore to obtain a basic condensation pH closest to physiological conditions, preferably of the order of a pH of 8.

The condensation agent is advantageously selected to have chemical groups favorable to the formation of non-covalent interactions such as, for example, electrostatic bonds and/or hydrogen bonds. In this way, the condensing agent assists in a controlled manner the polymerization of the silica monomers. The condensing agent is optimally a bio-inspired or natural or bio-sourced polymer.

The condensation agent is potentially recycled at the end of the process according to the invention. Recycling the condensation agent is an undeniable advantage. It is for example carried out by methods known to those skilled in the art such as by filtration techniques on exclusion gel and/or ion exchange resins, ultrafiltration on membranes of suitable nominal molecular weight limit.

Advantageously, the bile acid or its salt is a pore-forming structuring agent while being a natural surfactant.

Preferably, the bile acid is chosen from cholic acid, taurocholic acid, glycocholic acid, ursocholic acid, glycoursocholic acid, hyocholic acid, glycohyocholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, hyodeoxycholic acid, glycohyodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid or one of their salts, such as for example the ammonium salt or sodium. The bile salt can be adapted to the application of the porous silica produced.

The addition of bile acid or its salt makes it possible in particular to increase the carrying properties of the particles by creating pores and therefore by increasing the specific surface.

According to one possibility, the bile acid or its salt is added in step b) at a concentration less than or equal to 10 times its Critical Micellar Concentration (CMC). More precisely, the amount of bile acid added is between 0.05 and 10 times the CMC, more preferably, from 0.1 to 10 times the CMC.

Alternatively, the amount of bile acid is less than the CMC, preferably 0.05, more preferably 0.1 to 1 times the CMC.

Alternatively, the amount of bile acid is greater than the CMC, preferably up to 10 times the CMC.

The choice of concentration to use depends on the objective in terms of particle size. Indeed, this selection according to the invention has the advantage of modulating the size of the particles produced. Generally, the lower the concentration, the smaller the particle size.

Even when used under its CMC, bile acid, or one of its salts, can locally reach a sufficient concentration of aggregation to organize itself into supramolecular structures favoring the formation of pores within the silica produced.

The Critical Micellar Concentration or CMC is the surfactant concentration in a medium above which micelles form spontaneously. The CMC of a surfactant can be determined by techniques such as surface tension or light scattering measurements or else by potentiometric methods. For example, a measurement of the CMC of bile salts is given in the publication by Roda et al, journal of lipid research vol 31, 1990 1433

By way of preferred example, the bile acids or their salts are added to reach a final molar concentration in the matrix of less than or equal to 100 mM, preferably less than or equal to 60 mM, more preferably between 1 and 20 mM.

According to one embodiment, the at least one monovalent, divalent, or trivalent anion is chosen from at least one of a phosphate salt, a citrate salt, a tartrate salt, a sulfate salt, or a salt of nitrate.

By way of example, the phosphate salt is chosen from sodium phosphate, magnesium phosphate, potassium phosphate, calcium phosphate.

By way of example, the citrate salt is chosen from sodium citrate, potassium citrate, calcium citrate, magnesium citrate.

By way of example, the tartrate salt is chosen from sodium tartrate, potassium tartrate, calcium tartrate, sodium and potassium tartrate, choline tartrate, ammonium tartrate.

Preferably, the monovalent, bivalent or trivalent anion is added during step b) to reach a final concentration less than or equal to 300 mM, more precisely 200 mM, for example 60 mM. The addition of at least one anion or a mixture of anions in this concentration selection makes it possible to obtain the effect of the anion while limiting the impact on the pH of the condensation medium.

The at least one anion, or mixture of anions, monovalent, divalent or trivalent is added to the mixture of the condensing agent and the bile acid or its salt in order in particular to ensure electrostatic interactions at the level of the matrix, to control the formation of particles.

According to one possibility, the nuclei obtained in step a), also called hydrolysis phase, are added to the matrix obtained in step b) is also called matrix phase. According to one embodiment, the hydrolysis phase is added in a single portion to the matrix phase.

According to one embodiment, the method comprises a step c) of condensation in a basic medium. The condensation step condenses the silica nuclei obtained in step a) in a controlled manner thanks to the matrix obtained in step b). step condensation allows the nuclei obtained in step a) to grow in size to obtain the desired particles.

Step c) advantageously comprises mixing the silica nuclei and the matrix, preferably with stirring.

Advantageously, step c) of condensation is carried out at basic pH, said to be moderate, that is to say less than or equal to 10. Preferably, at pH less than or equal to 9, even more preferably less than or equal to 8 and greater than 7.

According to an advantageous embodiment of the present invention, the synthesis process is carried out at ambient temperature. Advantageously, the various steps a), b) and c) are carried out at ambient temperature, which advantageously does not require any heating or cooling. Advantageously, at least steps a), b) and c) of the synthesis process are carried out without heating or cooling. These arrangements ensure a process which consumes little energy and which allows synthesis under so-called mild conditions. The ambient temperature preferably means between 18°C and 30°C, more preferably between 20°C and 25°C.

According to one embodiment, the method according to the invention ensuring the synthesis of nanometric or submicron particles of porous silica advantageously comprises a step c′), subsequent to or simultaneous with step c) of condensation, comprising a step of functionalizing the particles . In the case where step c′) is subsequent to step c), it is advantageously directly subsequent, that is to say that step c) and step c′) are successive, preferably without step intermediate. The functionalization step makes it possible to modify the surface of the silica particles to lead to a targeted surface functionalization and to attribute new properties to the particles. The functionalization step advantageously comprising the addition of a silica precursor. According to one example, the silica precursor is chosen from at least one of TEOS (tetraethyl orthosilicate), TMOS (tetramethyl orthosilicate), MTMOS (methyltrimethoxysilane), MTEOS (methyltriethoxysilane), ETEOS (ethyltriethoxysilane), ETMOS (ethyltrimethoxysilane) , APTES ((3-Aminopropyl)triethoxysilane) or sodium orthosilicate or sodium metasilicate or an extract of biogenic silica such as for example from residues of rice or diatoms, sodium silicate or a natural source of ortho silicic acid. The silica precursor from the functionalization step can be identical to or different from the silica precursor used in step a).

According to one embodiment, the process according to the invention ensuring the synthesis of nanometric or submicron particles of porous silica advantageously comprises a step d), subsequent to step c) of condensation, preferably subsequent to step c′) if present, comprising a step of separating the particles. The separation step makes it possible to dissociate the particles from the matrix and from the nuclei that have not condensed. The step of separating the particles can, for example, be carried out by centrifugation or by tangential cross-flow filtration.

According to one embodiment, the method according to the invention ensuring the synthesis of nanometric or submicron particles of porous silica advantageously comprises a step of drying the particles e), after step c), preferably after step c' ) if present, and possibly in step d). The drying step makes it possible to obtain the particles in the form of a dry powder, which can be an advantage in terms of storage, for example. The step of drying the particles can for example be carried out by atomization or spray-drying. Advantageously, this step of drying by atomization or spray drying can lead to a step of mechanical shaping of the porous silica obtained.

According to one embodiment, the method according to the invention ensuring the synthesis of nanometric or submicron particles of porous silica advantageously comprises a step f), subsequent to step c), preferentially subsequent to step c′) if present, or optionally in step d) or in step e), of purification of the separated particles. The purification step is intended to allow the removal of organic residues resulting from the process, which is an advantage. However, due to the synthesis process according to the invention carried out under mild chemical conditions and in particular advantageously without organic solvent, any organic residues are not harmful and do not impact the properties of the particles. For example, bile salts do not have to be removed from the particles. By way of example, the purification step can be carried out by cycles of washing/centrifugation, or calcination.

By way of example, the purification phase by washing/centrifugation cycles is carried out by hot extraction in water comprising a dilute base such as for example an aqueous solution of ammonia, or ammonium hydroxide . For example, a treatment at a minimum of 85°C, advantageously for 2 hours, in a solution containing at least 1%, by weight, of ammonium hydroxide.

The purification phase is also referred to as the phase of elimination, in particular of bile salts. According to an advantageous possibility, the bile salts can thus be recycled.

Without being bound to a particular theory, it has been observed that the size of the particles increases with the concentration of silica precursor and of monovalent, bivalent or trivalent anion or and/or also the concentration of structuring agent: bile salts.

According to one aspect, the method comprises a step subsequent to step c), preferably to step d), and optionally to step e) or f), intended for the modification of the surface of the porous silica and more preferentially porous silica particles. The surface modification step makes it possible to modulate the surface properties such as the charge and/or the type of functions or ligands in order to carry out active targeting or for diagnosis. The surface modification step is carried out by chemical or thermal treatment.

Example 1: formation of porous silica particles in the presence of cholic acid.

A mixture containing 300 mM of MTMOS (methyltrimethoxysilane) acidified to pH 3, 15 g/L of polyethylenimine (25 kDa), 6.7 mM of cholic acid (in the form of ammonium salt, pH 9.5) and 5 mM of a pH 7 sodium phosphate solution is stirred at room temperature for about 45 min. The particles obtained are isolated by centrifugation (min 10000 rpm) and rinsed by washing with ultrapure water. The extraction of cholic acid is carried out by treatment at 85°C (minimum), 1 hour in a solution of 1% (by mass) minimum in ammonium hydroxide (figure 1A) or in a saturated solution of bicarbonate of sodium (Figure 1B). The particles were obtained with a yield of 73% and have an average diameter of 72 nm +/- 24 nm measured from observations by transmission electron microscopy (TEM) and reprocessed by the ImageJ software. The spherical morphology is checked by TEM (Figure 1). A specific surface of approximately 86 m ² /g was measured by absorption and desorption of gas (nitrogen) with the BET model (Figure 2). The particles exhibit a zeta potential of 32.1 mV and a hydrodynamic diameter measured by dynamic light scattering (DLS) of 177.5 nm (Figure 3). After a calcination step of 2 h at 550°C (ramp 5°C/min up to 550°C then maintained for 2 h at 550°C), the zeta potential is -19 mV and the primary diameter measured from of 61 nm +/- 14 nm TEM observations (Figure 1C).

Example 2: the formation of porous silica particles in the presence of cholic acid.

A mixture containing 300 mM of MTMOS acidified to pH 3, 15 g/L of polyethylenimine (25 kDa), 6.7 mM of cholic acid (in the form of ammonium salt, pH 9.5) and 30 mM of a solution of sodium phosphate pH 7 is stirred at room temperature for approximately 45 min. The particles obtained are isolated by centrifugation (min 10000 rpm), rinsed by washing with water and dried in a convection oven at 110°C (1 hour minimum). The particles were obtained with a yield of 51% and an average diameter of 100 nm +/- 19 nm measured from TEM observations. After a calcination step of 2 h at 550°C (5°C/min ramp up to 550°C then maintained for 2 h at 550°C), the spherical morphology was confirmed by SEM (Figures 4A and 4B respectively under magnification of x50000 and x10000). The presence of residual cholic acid before calcination was determined as being less than 5% of the total mass by thermogravimetric analysis (TGA) carried out with a ramp of 10° C./min from 25 to 800° C. (FIG. 5).

Example 3: formation of porous silica particles in the presence of deoxycholic acid

A mixture containing 300 mM of TMOMS acidified at pH 3, 15 g/L of polyethylenimine (25 kDa), 6.7 mM of sodium deoxycholate and 5 mM of a solution of sodium phosphate pH 7 is stirred at ambient temperature for approximately 45 mins. The particles obtained are isolated by centrifugation (min 10000 rpm) and rinsed by washing with water. Partial extraction of cholic acid is carried out by treatment at 85°C (minimum) for 2 hours in a solution containing at least 1% (by mass) of ammonium hydroxide. The particles were obtained with a yield of 70% and an average diameter of 293 nm +/- 83 nm measured by TEM (Figure 6A before, Figure 6B after, heat treatment in ammonia).

Example 4: formation of porous silica particles in the presence of taurocholic acid

A mixture containing 300 mM of TMOMS acidified to pH 3, 15 g/L of polyethylenimine (25 kDa), 6.7 mM of sodium taurocholate (in the form of ammonium salt, pH 9.5) and 5 mM of a sodium phosphate solution pH 7 is stirred at room temperature for about 45 min. The particles obtained are isolated by centrifugation (min 10000 rpm) and rinsed by washing with water. Partial extraction of cholic acid is carried out by treatment at 85°C (minimum) for 2 hours in a solution containing at least 1% (by mass) of ammonium hydroxide. The particles were obtained with a minimum yield of 50% and an average diameter of 65 nm +/- 10 nm measured by TEM (Figure 7 A before, Figure 7B after, heat treatment in ammonia).

Example 5: formation and functionalization of porous silica particles in the presence of a mixture of orqanosilane and cholic acid

A solution containing 300 mM of MTMOS acidified at pH 3, 15 g/L of polyethylenimine (25 kDa), 6.7 mM of cholic acid (in the form of ammonium salt, pH 9.5), 5 mM of a solution of sodium phosphate pH 7 and for the functionalization 15 mM (example 5A) or 30 mM (Example 5B) of APTES (3-aminopropyl)triethoxysilane) stirred at room temperature for about 45 min. In Example 5C, APTES in an amount of 15 mM is added after 20 minutes of stirring. In Example 5D, APTES in an amount of 30 mM is added after 20 minutes of stirring. The particles obtained are isolated by centrifugation (min 10000 rpm) and rinsed by washing with water. The extraction of the cholic acid is carried out by treatment at 85° C. (minimum), 1 hour in a solution containing at least 1% (by mass) of ammonium hydroxide. The particles were obtained with a minimum yield of 50% and an average diameter of less than 100 nm measured by TEM (Table 1). The spherical morphology is checked by transmission microscopy (TEM) (FIGS. 8A to 8D). The particles have a zeta potential above +20 mV and a hydrodynamic diameter measured by DLS below 190 nm.

Table 1: Primary and hydrodynamic diameters of silica particles and their zeta potential (examples 5A to 5D)

Example 6 Formation of Porous Silica Particles in the Presence of Deoxycholic Acid A mixture containing 300 mM of TMOMS acidified to pH 3, 15 g/L of polyethylenimine (25 kDa), 3.3 mM of sodium deoxycholate and 5 mM of a solution of sodium phosphate pH 7 is stirred at room temperature for approximately 45 min. The particles obtained are isolated by centrifugation (min 10000 rpm) and rinsed by washing with water. Partial extraction of cholic acid is carried out by treatment at a minimum of 70°C for 2 hours in a solution containing at least 1% (by mass) of ammonium hydroxide.

The particles were obtained with a minimum yield of 50% and an average diameter of 123 nm +/- 25 nm measured by TEM (FIG. 9 after, heat treatment in ammonia). Example 7: formation of porous silica particles in the presence of deoxycholic acid

A mixture containing 300 mM of TMOMS acidified at pH 3, 15 g/L of polyethylenimine (25 kDa), 1.7 mM of sodium deoxycholate and 5 mM of a solution of sodium phosphate pH 7 is stirred at ambient temperature for approximately 45 mins. The particles obtained are isolated by centrifugation (min 10000 rpm) and rinsed by washing with water. Partial extraction of cholic acid is carried out by treatment at 70°C (minimum) for 2 hours in a solution containing at least 1% (by mass) of ammonium hydroxide. The particles were obtained with a minimum yield of 50% and an average diameter of 63 nm +/- 12 nm measured by TEM (Figure 10 after, heat treatment in ammonia).

The invention is not limited to the embodiments described above and extends to all the embodiments covered by the claims.

Claims

1. Process for the synthesis of nanometric or submicron porous silica particles comprising the following steps: a. preparation of silica nuclei by hydrolysis of at least one silica precursor in water in an acid-catalyzed medium, the quantity of acid is chosen so as to be less than or equal to 0.1 equivalent of acid relative to the silica precursor, then b. formation of a matrix phase by mixing at least one condensation agent chosen from a basic polycationic branched or linear polymer, and at least one bile acid, or its salt, then adding at least one monovalent, divalent anion or trivalent selected from a phosphate salt, a citrate salt, a tartrate salt, a sulfate salt, or a nitrate salt, to the mixture comprising the condensing agent and bile acid or its salt, c. condensation in a basic medium of the silica nuclei obtained in step a) by mixing, with stirring, the silica nuclei obtained in step a) and the matrix phase obtained in step b) at an alkaline pH less than or equal to 10 .

2. Method according to the preceding claim, in which steps a) to c) are carried out at room temperature.

3. Method according to any one of the preceding claims, in which the bile acid is chosen from cholic acid, taurocholic acid, glycocholic acid, ursocholic acid, glycoursocholic acid, hyocholic acid, glycohyocholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, hyodeoxycholic acid, glycohyodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid or a salt thereof.

4. Method according to any one of the preceding claims, in which the silica precursor is chosen from at least one of TEOS (tetraethyl orthosilicate), TMOS (tetramethyl orthosilicate), MTMOS (methyltrimethoxysilane), MTEOS (methyltriethoxysilane), ETEOS (ethyltriethoxysilane), ETMOS (ethyltrimethoxysilane), APTES ((3-Aminopropyl)triethoxysilane), sodium orthosilicate or sodium metasilicate.

5. Process according to any one of claims 1 to 3, in which the silica precursor is chosen from at least one of a biogenic silica extract, sodium silicate or a natural source of ortho silicic acid.

6. Method according to any one of the preceding claims, in which in step a) the silica precursor is in an amount less than or equal to 3000 mM.

7. Process according to any one of the preceding claims, in which the condensation agent is chosen from polyamino acids, polyallylamines, polyethylene imines, derivatives of propylene imines

8. Process according to any one of the preceding claims, in which the condensation agent is used in an amount less than or equal to 100 g/L, preferably less than or equal to 50 g/L, preferably less than or equal to 20 g/L.

9. Method according to any one of the preceding claims, in which the bile acid or its salt is added in an amount less than or equal to 10 times the Critical Micellar Concentration (CMC), preferably between 0.05 and 10 times the CMC, more precisely between 0.1 and 10 times the CMC.

10. Method according to any one of the preceding claims, in which the at least one monovalent, divalent or trivalent anion is added in a concentration of less than or equal to 200 mM.

11. Process according to any one of the preceding claims, comprising a step c′) of functionalizing porous silica particles obtained in step c) by adding a silica precursor, step c′) being simultaneous or successive in step c).

12. Process according to any one of the preceding claims, comprising, after step c), a step d) of separating the particles and optionally a step e) of drying the porous silica particles obtained and optionally a step f) of purifying the particles isolated.

13. Process according to the preceding claim, in which step d) of separation is carried out by centrifugation or filtration by tangential route.

14. Method according to any one of the two preceding claims, in which step e) of drying is carried out by atomization.

15. Process according to any one of the three preceding claims, in which step f) of purification to eliminate organic residues is carried out by washing, chemical extraction or calcination.