EP2498761A1 - Silica particle including a molecule of interest, method for preparing same and uses thereof - Google Patents

Abstract

The present invention relates to a porous nanoparticle of silica, including at least one molecule of interest, the silica lattice inside said nanoparticle being functionalized by means of at least one grouping capable of establishing a non-covalent ionic and/or hydrogen bond with the molecule of interest, by means of which the molecule(s) of interest is (are) bonded to the silica lattice solely by non-covalent bonds. The present invention also relates to a method for preparing such a silica particle and to the uses thereof.

Description

 SILICA PARTICLE INCORPORATING A MOLECULE OF INTEREST, ITS PREPARATION METHOD AND ITS USES

DESCRIPTION

TECHNICAL AREA

 The present invention relates to the field of silica particles and, more particularly, silica particles capable of incorporating, encapsulating or confining molecules of interest such as biomolecules.

 The present invention therefore proposes a silica particle incorporating, encapsulating or confining a molecule of interest. In the context of the present invention, advantage is taken of the presence, within the silica particle, of groups capable of establishing a non-covalent bond, of the electrostatic interaction and / or hydrogen bridge type, with said molecule of interest .

 The present invention also relates to a process for preparing such a silica particle ie a process for incorporating, encapsulating or confining a molecule of interest in a silica particle and the uses of such a particle, in particular in the fight against counterfeiting or in biological analysis.

STATE OF THE PRIOR ART

The synthesis of silica nanoparticles by sol-gel using silicon alkoxides is described in the literature since the work of Stôber in the late 1960s which developed the a process bearing his name which makes it possible to obtain micrometric silica particles [1].

 From this process, numerous developments have made it possible to reduce the size of the particles to obtain nanoparticles ([2],

[3]). The Stöber synthesis method uses a silicon alkoxide such as tetraethoxysilane (TEOS) or tetramethoxysilane (TMOS) in a solution of alcohol and water. The solution is then heated in order to carry out the hydrolysis of the silicon alkoxide, and then the addition of a certain amount of catalyst, in this case HCl, allows the condensation of the silica in the form of particles. Another mode of synthesis based on a sol-gel reaction within an emulsion makes it possible to obtain particles of smaller size and more monodisperse than with the Stöber process. This process is called sol-gel synthesis by inverse microemulsion ([4] - [6]).

 In this process, an emulsion of water in an oily phase is formed using a surfactant (or surfactant) and in some cases a co-surfactant (or co-surfactant). The size of the micelles is generally nanometric, they thus form a nanoreactor in which the hydrolysis-condensation reaction of the silicon alkoxide occurs. It is the size of the micelle that determines the size of the formed particle. In the case of the Stôber method,

Encapsulation of an organic molecule requires a covalent grafting between the latter and the silica network. Thus, by this synthetic route, a number of organic fluorophores have been incorporated in the silica particles. For this, it is necessary to create a silicon alkoxide covalently bound to the organic molecule by reaction between an isothiocyanate function and an amine function. Numerous examples are given in the literature with FITC (for fluorescein isothiocyanate) or RBITC (for Rhodamine B isothiocyanate) encapsulated in silica by the Stöber process [7].

 In these processes, the fluorophore having an isothiocyanate function is mixed with aminopropyltriethoxysilane (APTES). The isothiocyanate function reacts with the amine function of the alkoxide and forms a fluorescent silicon alkoxide which is then introduced into the synthesis in a certain proportion relative to the main silicon alkoxide TEOS. In general, this concentration is of the order of 1 to 5%. However, this concentration can be higher and reach up to 25%. When the fluorescent alkoxide obtained by reaction between the isothiocyanate function and the APTES is introduced at the beginning of the reaction, the fluorophore is distributed throughout the particle.

This pathway has also been used to form heart / shell type structures with either the core comprising the organic molecule and the surface of the silica is inversely [8]. In this case, the process is carried out in two identical successive steps.

This covalent graft encapsulation method also works for non-fluorescent molecules using the same procedure: reaction between a silicon alkoxide (APTES) and the molecule to be covalently bonded with the silica ([9], [10]) . It is also possible to obtain a silica core and a shell containing the organic molecules by covalent grafting or by sol-gel reaction ([7], [11],

[12]).

In the case of micellar syntheses, the method of encapsulation of organic molecules by covalent grafting as for the Stöber method previously described also works but is not necessarily necessary. Indeed, being in a micellar medium makes it possible to confine molecules or particles within the micelle and thus to encapsulate them with silica.

Research carried out in the Applicant's laboratory has shown that this process makes it possible to encapsulate fluorescent organic molecules such as rhodamine and fluorescein [13]. This encapsulation process works for soluble molecules in the aqueous phase. In this process, after the formation of the inverse emulsion, the first step is to introduce the molecule to be encapsulated which, if it has a higher solubility in water than in the phase oily, will concentrate primarily inside the micelles. Then the silicon alkoxide (TEOS) and catalyst for the formation of the silica particles are introduced. The formation of the silica particle is from the outside of the micelle inward thus encapsulating the molecule present in the micelle. The silica particles thus obtained are then functionalized by addition of coupling agents such as N-2- (aminoethyl) -3-aminopropylthiethoxysilane.

 Work carried out in the Applicant's laboratory has shown that depending on the solubility of the organic molecule, it will, if it is very soluble in water, be found confined to the heart of the silica particle. In the case where its solubility is lower, it will either be distributed in the particle or be more on the surface. If it is not soluble in water at all and does not form a covalent bond with silica, it is not incorporated in the particle.

In the case of deoxyribonucleotide acid (DNA) and biological macromolecules, strategies and methods of encapsulation have been described in the literature. Biomacromolecules have already been successfully encapsulated in micrometric and nano-sized objects dispersed in water (hydrophilic objects).

Biomacromolecules such as bovine serum albumin (BSA) and DNA have been successfully encapsulated in hollow silica microcapsules [14]. These microcapsules are synthesized by double micellar pathway (water-oil-water). The objects obtained have a micrometric size. The same encapsulation format was developed by the classical sol-gel method, the Stöber method. The presence of DNA in the objects was revealed by fluorescence using ethidium bromide (BET) type DNA intercalants. A similar method described in the literature relates the synthesis of sol-gel silica nanoparticles with smaller diameters ~ 200 nm [15].

Different silica nanoparticles (~ 40 nm) have been synthesized using an inverse microemulsion using TEOS, in which, in the core part of these nanoparticles, there are biomolecules conjugated with the Cy5 fluorophore (cyanine which fluoresces in the red). 650-670 nm) [16]. The molecules studied and conjugated with Cy5 are polylysine (70000-15000 g.mol ^-1 ), immunoglobulin G (IgG, 150000 g, mol- ¹ ), BSA (68000 g, mol- ¹ ), insulin (5700 g mol ^-1 ) and pepsin (42500 g mol ^-1 ). The presence of the biomolecular core was revealed by the fluorescence emitted by Cy5. In addition, it is important to emphasize that these biomolecules have characteristics of weight and size lower than those of the plasmid DNA used in the present invention ie DNA consisting of 3000 base pairs (bp). The use of a micellar synthesis method for the encapsulation of DNA in polymer particles has already been published [17]. The article by Hammady et al. gives by way of example the encapsulation in PVA beads

(polyvinylacrylic) and PLA (polylactide) DNA. In this case, the DNA is a priori inside the beads. However, it is neither accessible for analysis nor usable until the polymer is destroyed by dissolution. Consequently, the difference with respect to the present invention described below concerns, on the one hand, the material encapsulating the DNA (polymer) and, on the other hand, the need to dissolve it to gain access to the DNA.

There may also be other types of particles or "containers" encapsulating DNA. The article by Cisse et al. describes the encapsulation of DNA in capsules whose wall is formed of a lipid membrane [18]. This consists of a double layer of surfactant. This type of particle does not have much to do with a silica particle according to the present invention since they are in a way bubbles, with a liquid inside, composed of a thin wall formed by a double molecular layer. . A third approach is to functionalize the surface of nano-objects in order to anchor the desired biomolecules ([19], [20]). In these documents, the steps of preparation and functionalization of the nanoparticles are distinct and carried out prior to contact with the biomolecules. Generally, the surface of nano-objects is functionalized by amino groups which, by electrostatic interactions, bind with biomolecules. The nanoparticles have a positive surface charge thanks to the amino groups. Thus, DNA enrichment is carried out in the form of "nanoparticle-DNA" complexes that involve electrostatic bonds between the amino groups of the nanoparticles and the negative charge of the phosphate groups of the DNA. Similar procedures have been developed to improve complexation with DNA. Thus, a regular porosity has been generated in silica nanoparticles in order to better accommodate the proximity of the biomolecule and to promote the electrostatic bonding with the functionalized surface of the silica nanoparticles [21].

A final route is used to encapsulate DNA or biomolecules in silica sol-gel film form. This way does not present particles but films. Biomolecules have been stored in inorganic or organic-inorganic hybrid gels. DNA encapsulation (50 bp) was performed in hybrid polyvinyl alcohol-silica gels. Analysis of the small gel-encapsulated DNA fragments showed that the complexation between the silica network and the phosphate groups probably occurs. This explains why most of the DNA molecules could not be extracted from the gels [22]. There is therefore a real need for silica particles such as silica nanoparticles capable of incorporating or encapsulating a molecule of interest and especially a molecule of interest of large size such as DNA, to protect the molecule of interest while leaving it accessible to the elements and constituents of the external environment. In addition, such particles must be able to be prepared by an easy to implement process that does not require the preparation of reaction precursors.

STATEMENT OF THE INVENTION

The present invention overcomes the disadvantages and technical problems listed above. Indeed, the latter proposes particulate materials, spherical silica-based and in particular nanoparticulate, porous, incorporating molecules of interest in which the molecules of interest are maintained in the particles while remaining accessible to elements present in the external particles such as enzymes and without implementation of any covalent bond between the molecules of interest and the particles. Despite their confinement, the molecules of interest retain their chemical reactivity. As a result, molecules present on the outside of the particles can access, through the porosity of the particles, the molecules of interest and react with them. In addition, such materials can be prepared by an industrially applicable process, not requiring procedures or heavy steps and using easily accessible products.

 The work of the inventors has demonstrated that the combined use of a silicon alkoxide, also called "silane compound", forming a complete network and a silicon alkoxide having at least one group capable of establishing a Non-covalent binding with a molecule of interest makes it possible to manufacture silica particles such as silica nanoparticles incorporating or confining molecules of interest. Indeed, the silica particles obtained by such a process are functionalized within the silica network by groups capable of establishing a non-covalent bond. This specific functionalization allows non-covalent bonds of the electrostatic bond or hydrogen bridge type with the molecule of interest.

Thus, by way of example, when the molecule of interest is DNA, the latter compound of sugar-bound phosphates which are themselves linked to the nitrogenous bases has a negative zeta potential. The use of a silicon alkoxide having one or more amino group (s) and a silicon alkoxide forming a complete network makes it possible to obtain silica particles functionalized with amino groups within the silica network. The negatively charged phosphate interacts electrostatically (or by hydrogen bridge) with the amino groups of the silica network. During the hydrolysis of the silicone derivatives ie alkoxides of silicon, the DNA is found confined in the silica particle and in particular in the core of the silica particle which is mainly due to the electrostatic bonds and the hydrophilic properties of the DNA. The DNA is then encapsulated in the porous silica network of the particles and in particular in the center of such a network. A schematic diagram of the final structure of the particle is shown in Figure 1.

It should be noted that the silica particles according to the present invention are not hollow particles. These particles may appear as heart / shell type particles, especially when analyzed by transmission electron microscopy. These particles are not, however, "real" particle type heart / shell since they do not have a heart with the ^era chemical composition and a shell with a 2 ^nd chemical composition, both compositions are distinct and do nor do they present an interface between the heart and the materialized shell. However, for the sake of brevity, we use in the present invention the term "heart" to define the center of the particle comprising almost all the molecules of interest and the term "shell" to define the outer portion of the particle does not comprise a molecule of interest.

The encapsulated molecules of interest are protected by silica. The silica thus serves as a stabilizing support for the molecule of interest. However, the process according to the invention makes it possible to obtain a silica shell surrounding the core of the particle containing the molecule of interest. This shell is sufficiently porous to have accessibility to the molecule of interest of the constituents present in the external medium and this, both for small molecules such as fluorescent nucleotides, and for larger molecules such as enzymes.

 In addition, since the encapsulation according to the invention involves non-covalent bonds, the properties of the molecule of interest such as activity and / or recognition are not modified.

The present invention thus relates to a porous silica particle, incorporating at least one molecule of interest and essentially obtained from hydrolysis:

at least one first silicon alkoxide of the formulas Si (ORi) ₄ , R ₂ Si (OR ₃ ) ₃ or R ₄ RsSi (OR ₆ ) 2 in which R 1, R 3 and R 6, which may be identical or different, represent an alkyl radical from 1 to 6 carbon atoms and R ₂ , R 4 and R ₅ , which may be identical or different, represent a hydrogen, an alkyl radical of 1 to 6 carbon atoms or an alkenyl radical of 1 to 6 carbon atoms, and

at least one second silicon alkoxide having at least one group capable of establishing a non-covalent ionic bond and / or hydrogen with the molecule of interest. More particularly, the present invention relates to a silica particle comprising at least a molecule of interest, the silica network inside said particle being functionalized by at least one group capable of establishing a non-covalent ionic bond and / or hydrogen with the molecule of interest, whereby the (or) molecule (s) of interest is (are) bound to the silica network only by non-covalent bonds.

 Advantageously, the silica particle according to the invention has a silica network functionalized by several groups capable of establishing a non-covalent ionic bond and / or hydrogen with the molecule of interest. These groups are distributed in the silica particle in the form of a decreasing gradient from the center of the particle towards the outside of the particle, no group being present on the surface of the particle.

By "silica particle incorporating at least one molecule of interest" is meant, in the present invention, a silica particle within which there is at least one molecule of interest. In the context of the present invention, the molecule of interest or all the molecules of interest are not on the surface of the silica particle.

Advantageously, the silica particle according to the invention is a core / shell particle in the core of which is at least one molecule of interest. When the silica particle incorporates several molecules of interest, identical or different, the latter are found Majority inside the particle and especially in the heart of the particle.

 The distribution of the molecules of interest in the silica particle may be in the form of a gradient with a high concentration of molecules of interest in the center of the particle and in particular in the center of the core of this particle and a lower concentration at as we move away from this center.

 Thus, at least 80%, at least 90%, at least

95%, at least 98%, at least 99% of the molecules of interest and / or all the molecules of interest are found inside and especially in the core of the particle. The terms "silica particle encapsulating at least one molecule of interest", "silica particle incorporating at least one molecule of interest" or "silica particle confining at least one molecule of interest" are equivalent and can be used interchangeably .

The silica particle according to the invention is of nanometric size; we can therefore speak of "nanoparticle", "ball" or "nanobille". As presented in the experimental part below, it is possible to vary the average size of the silica particles according to the invention by varying the amounts of molecules of interest and / or the polar liquid ratio (polar solvent such as water) / surfactant / non-polar liquid (oily phase consisting mainly of the non-polar or weakly polar solvent). Advantageously, the silica particles according to the invention an average size of less than or equal to 150 nm, 120 nm, 100 nm, 80 nm or 60 nm. The silica particles according to the invention may have an average size of the order of 40 nm (ie 40 ± 10 nm). In a variant, the silica particles according to the invention have an average size of less than 40 nm, at 20 nm and in particular of the order of 10 nm (ie 10 + 5 nm). It is clear to those skilled in the art that the average size of the silica particle according to the invention will influence the size of the molecule (s) of interest incorporated (s).

The silica particles according to the invention are porous with open porosity and especially mesoporous with open porosity. Advantageously, they have a pore size of less than 100 angstroms and a pore size distribution ranging from 1 to 100 angstroms, in particular from 10 to 90 angstroms, in particular from 15 to 80 angstroms and, especially, from 20 to 70 angstroms. angstroms and a surface area of 200 to 900 m ² . g ^-1 , in particular from 300 to 800 m ² . g ^-1 and, in particular, from 400 to 700 m ² . g ^-1 .

 The silica particle according to the invention has a number of characteristics listed below:

 the molecule of interest is in the silica particle and in particular in the core of this particle, the silica plays a protective role vis-à-vis the molecule of interest,

the molecule of interest is maintained in the silica particle by virtue of the non-covalent bonds implemented, no covalent bond existing between the silica network and the molecule of interest;

 the silica particle being porous, small molecules can enter the silica particle (when the molecule of interest is a nucleic acid, an intercalant can be interposed in the nucleic acid and emit a specific signal in the presence of this molecule. acid),

 the molecule of interest incorporated in the silica particle is also accessible to enzymatic reactions of which it is the substrate, such enzymatic reactions being able to cause a detectable signal such as a modification of the labeling of the molecule of interest.

 Thus, the molecule (s) of interest incorporated in the silica particle according to the invention is (are) maintained and protected (s) in the silica particle according to the invention. The molecule (s) of interest incorporated into the silica particle according to the invention is (are) accessible to molecules present in the external medium of the silica particle according to the invention. . The molecule (s) of interest incorporated into the silica particle according to the invention is (are) capable of interacting with these molecules.

By "molecule of interest" is meant in the context of the present invention any molecule having at least one group capable of establishing a non-covalent ionic bond and / or hydrogen with the second silicon alkoxide as defined in the present invention. present invention. This molecule can be natural or synthetic, small or large (macromolecule). The molecule of interest is advantageously chosen from the group consisting of an enzyme, a protein, an oligopeptide, a peptide, an antigen, an antibody, a nucleic acid, a polymer or a carbohydrate. The molecule of interest may be labeled in particular by a fluorochrome (fluorescein, Cy5, Cy3, rhodamine), a radioactive isotope, an enzyme (alkaline phosphatase, horseradish peroxidase), colloidal gold, biotin or digoxygenine.

 The term "nucleic acid" as used herein is equivalent to the following terms and expressions: "polynucleotide sequence", "nucleotide molecule", "polynucleotide", "nucleotide sequence". By "nucleic acid" is meant, in the context of the present invention, a chromosome; a gene ; a regulatory polynucleotide; DNA, single-stranded or double-stranded, genomic, chromosomal, chloroplast, plasmidic, mitochondrial, recombinant or complementary; total RNA; messenger RNA; ribosomal RNA; a transfer RNA; a portion or a fragment thereof.

 A DNA in the context of the present invention may have from 10 bp (or 20 nucleotides) to 5,000 bp (or 10,000 nucleotides), and in particular from 20 bp (or 40 nucleotides) to 4,000 bp (or 8,000 nucleotides) .

In the context of the present invention, the term "non-ionic covalent bond" means an intermolecular interaction between at least two positively or negatively charged groups. The terms "non-ionic covalent bond", "ionic interaction", "electrostatic bond" or "electrostatic interaction" are equivalent and interchangeably usable.

 In the context of the present invention, this intermolecular interaction is attractive. It involves a negatively charged group of the molecule of interest and a positively charged group of the second silicon alkoxide. As a variant, it involves a positively charged group of the molecule of interest and a negatively charged group of the second silicon alkoxide. By "non-covalent hydrogen bond" is meant a bond in which a hydrogen atom covalently bonded to an A atom is attracted to a B atom containing a pair of free electrons (: B). This results in a strong polarization of the A-H bond and electrostatic interactions between Η (δ +) and: B. The term "non-covalent hydrogen bond" is equivalent to the expression "dipole-dipole interaction".

In the context of the present invention, the atom A may be an atom of the second silicon alkoxide and the atom B an atom of the molecule of interest. Alternatively, the atom A may be an atom of the molecule of interest and the atom B an atom of the second silicon alkoxide. In the context of the present invention, the first silicon alkoxide of formulas Si (ORi) 4, R ₂ Si (OR ₃ ) ₃ or R ₄ R ₅ Si (OR ₆ ) 2 in which R 1, R ₃ and R ₆ , which are identical or different, represent an alkyl radical of 1 to 6 carbon atoms and R ₂ , R ₄ and R 5, identical or different, represent a hydrogen, an alkyl radical of 1 to 6 carbon atoms or an alkenyl radical of 1 to 6 carbon atoms. carbon participates mainly in the formation of the silica network and in particular to the formation of the shell of the silica particle according to the invention.

 Indeed, this first silicon alkoxide may advantageously have a hydrolysis rate less than or equal to the rate of hydrolysis of the second silicon alkoxide.

 By "alkyl radical of 1 to 6 carbon atoms" is meant an alkyl radical, linear or branched, having from 1 to 6 carbon atoms and especially from 1 to 4 carbon atoms.

 By "alkenyl radical of 1 to 6 carbon atoms" is meant an alkenyl radical, linear or branched, having at least one double bond and 1 to 6 carbon atoms and especially 1 to 4 carbon atoms.

Advantageously, R ₂ , R ₄ and R ₅ , which are identical or different, are chosen from the group consisting of hydrogen, methyl, ethyl, vinyl and propyl.

The first silicon alkoxide that can be used in the context of the present invention is especially chosen from the group consisting of tetramethoxysilane (TMOS, Si (OCH ₃ ) ₄ ), tetraethoxysilane (TEOS, Si (OC ₂ H ₅ ) ₄ ), tetrapropoxysilane (TPOS, Si (OC ₃ H ₇ ) ₄ ), tetrabutoxysilane (TBOS, Si (OC ₄ H ₉ )), trimethoxysilane (TMOS, HSi (OCH ₃ ) ₃ ), methyltrimethoxysilane [(CH ₃ ) Si (OCH ₃ ) ₃ ], ethyltrimethoxysilane [(C ₂ H ₅ ) Si (OCH ₃ ) ₃ ], propyltrimethoxysilane [(C ₃ H ₇ ) Si (OCH ₃ ) ₃ ], vinyltrimethoxysilane [(O ₂ ¾) Si (OCH ₃ ) ₃ ], triethoxysilane [HSi (OC ₂ H ₅ ) ₃ ], methyltriethoxysilane [(CH ₃ ) Si (OC ₂ H ₅ ) ₃ ], ethyltriethoxysilane [( C ₂ H ₅ ) If (OC ₂ H ₅ ) ₃ ], propyltriethoxysilane

[(C ₃ H ₇ ) Si (OC ₂ H ₅ ) ₃ ], vinyltriethoxysilane

[(C ₂ H ₃ ) Si (OC ₂ H ₅ ) ₃ ], and mixtures thereof.

 Advantageously, the first silicon alkoxide used in the context of the present invention is the TMOS or the TEOS.

 More particularly, the first silicon alkoxide used in the context of the present invention is TEOS. Indeed, such a precursor makes it possible to obtain, for the silica shell, a layer whose porosity is optimal to allow accessibility to the molecule (s) of interest (s) such as DNA encapsulated in the core of the particle by large constituents present in the external medium.

The second silicon alkoxide used in the context of the present invention comprises at least one group capable of establishing a non-covalent ionic bond and / or hydrogen with the molecule of interest. This second alkoxide may have a rate of hydrolysis equal to or greater than the speed hydrolyzing the first silicon alkoxide thereby to concentrate the molecule (s) of interest in the heart of the silica particle.

 The absence of such an alkoxide prevents the encapsulation of the molecule of interest in the silica particle.

The second silicon alkoxide is advantageously of formulas R ₇ Si (ORs) 3 or RgRi oSi (ORn) 2 in which Rs and Ru, identical or different, represent an alkyl radical of 1 to 6 carbon atoms and R ₇ , R 9 and R 11, which may be identical or different, represent an alkyl radical of 1 to 8 carbon atoms, a heteroalkyl radical of 1 to 10 carbon atoms, an alkylaryl radical of 1 to 12 carbon atoms or an alkenyl radical of 1 to 8 carbon atoms. carbon atoms,

the radical R ₇ and at least one of the radicals R 9 and R 10 being substituted by at least one group capable of establishing a non-covalent ionic bond and / or hydrogen with the molecule of interest.

 The radicals Rg and Ru, identical or different, represent an alkyl radical of 1 to 6 carbon atoms as previously defined.

 By "alkyl radical of 1 to 8 carbon atoms" is meant an alkyl radical, linear or branched, having from 1 to 8 carbon atoms, especially from 1 to 6 carbon atoms and, in particular, from 1 to 4 atoms of carbon.

By "heteroalkyl radical of 1 to 10 carbon atoms" is meant a linear or branched alkyl radical having from 1 to 10 carbon atoms, in particular from 1 to 8 carbon atoms and, in particular, from 1 to 6 carbon atoms and having at least one heteroatom such as N, S, O or P.

 By "alkylaryl radical of 1 to 12 carbon atoms" is meant a linear or branched alkyl radical having from 1 to 12 carbon atoms, especially from 1 to 10 carbon atoms, in particular from 1 to 8 carbon atoms. carbon and, more particularly, from 1 to 6 carbon atoms, having an aromatic or heteroaromatic substituent of 3 to 8 carbon atoms and optionally at least one heteroatom such as N, S, O or P.

 By "alkenyl radical of 1 to 8 carbon atoms" is meant a linear or branched alkenyl radical having at least one double bond and from 1 to 8 carbon atoms, in particular from 1 to 6 carbon atoms, and in particular , from 1 to 4 carbon atoms.

By "group capable of establishing a non-covalent ionic bond and / or hydrogen" is meant in the context of the present invention a group selected from the group consisting of -N¾, -NHR ₁₂ with R ₁₂ representing an alkyl radical of 1 to 6 carbon atoms as previously defined, -NH ₃ ⁺ , -NH ₂ R 13 ⁺ with R 13 representing an alkyl radical of 1 to 6 carbon atoms as previously defined, -COOH, -COO ^~ , C (O) NH, -C (O), -SH and -OH. The group of the molecule of interest participating in the non-covalent bond with the function in the core of the particle derived from the second silicon alkoxide may also comprise such a group. Advantageously, the second silicon alkoxide that can be used in the context of the present invention is chosen from the group consisting of N-trimethoxysilylpropyl-N, N, N-trimethylammonium chloride.

(C ₉ H ₂₄ ClNO ₃ Si, CAS: 35141-36-7);

Aminoethylaminomethyl) phenethyltrimethoxysilane

(C ₄ H ₂₆ N ₂ O ₃ Si, CAS: 74113-77-2); N- (6-aminohexyl) aminopropyltrimethoxysilane (Ci ₂ H ₃ O ₂ O ₃ Si,

CAS: 51895-58-0); 3-aminopropyl-methyl-diethoxysilane (C ₈ H ₂ INO ₂ Si, CAS: 3179-76-8); 3-aminopropyl-trimethoxysilane (APTMES, C ₆ H ₇ N0 ₃ Si, CAS: 13822-56-5); 3-aminopropyltriethoxysilane (APTES, C ₉ H ₂₃ N0 ₃ Si, CAS: 919-30-2); 3- (2-aminoethylamino) propyl-trimethoxysilane

((CH ₃ 0) ₃ Sr (CH ₂ ) ₃ NHCH ₂ CH ₂ NH ₂ ), CAS: 1760-24-3); (3-Mercaptopropyl) trimethoxysilane (HS (CH ₂ ) ₃ Si (OCH ₃ ) ₃ , CAS: 4420-74-0), (3-mercaptopropyl) triethoxysilane (HS (CH ₂ ) ₃ Si (OC ₂ H ₅ 3, CAS: 14814-09-6) and mixtures thereof.

 The second silicon alkoxide that can be used in the context of the present invention is, more particularly, APTMES.

The silica particle according to the invention is essentially obtained from the hydrolysis of at least a first silicon alkoxide and at least one second silicon alkoxide as previously defined. The groups capable of establishing a non-covalent ionic bond and / or hydrogen with the molecule of interest functionalizing the silica network of the particle are the result of the hydrolysis of the second silicon alkoxide. The silica particle according to the invention consists essentially of units resulting from the hydrolysis of at least a first silicon alkoxide and at least one second silicon alkoxide as previously defined. Indeed, the silica particle may comprise other elements including at least one element capable of imparting magnetic properties to it. Such elements capable of imparting magnetic properties are chosen especially from the group consisting of iron, gadolinium, nickel, copper, chromium, cobalt, gold, silver, platinum, palladium, an oxide and a hydroxide thereof. Thus, the silica particle according to the invention but without the molecule (s) of interest is at least 80%, at least 90%, at least 95%, at least 98%, at least 99%. % units derived from the hydrolysis of at least a first silicon alkoxide and at least one second silicon alkoxide as previously defined. Alternatively, the silica particle according to the invention but without the molecule (s) of interest consists only of units resulting from the hydrolysis of a (or) first (s) alkoxide (s). ) silicon and a (or) second (s) alkoxide (s) of silicon as previously defined.

The present invention also relates to a process for preparing a silica particle incorporating at least one molecule of interest according to the present invention. Any method for preparing such a particle from at least a first silicon alkoxide and at least one second alkoxide of Silicon as previously defined can be used in the context of the present invention.

 Advantageously, the present invention relates to a process for the preparation, in the presence of a molecule of interest, of at least one silica particle by inverse emulsion from:

at least one first silicon alkoxide (e.e.) of the formulas Si (ORi) ₄ , R ₂ Si (OR ₃ ) ₃ or R ₄ R ₅ Si (OR ₆ ) ₂ ) as previously defined,

 - At least a second silicon alkoxide having at least one group capable of establishing a non-ionic covalent bond and / or hydrogen with the molecule of interest as defined above. By "microemulsion inverse" also called microemulsion "water in oil" means a clear suspension, thermodynamically stable, fine droplets of a first polar liquid in a second non-polar liquid and therefore immiscible with the first liquid. The terms "reverse micellar" and "reverse microemulsion" are equivalent and interchangeable.

The method according to the present invention can implement:

 a first silicon alkoxide as previously defined and a second silicon alkoxide as previously defined;

a first silicon alkoxide as previously defined and a plurality of second silicon alkoxides as previously defined; several first silicon alkoxides as previously defined and a second silicon alkoxide as previously defined; or

 several first silicon alkoxides as defined above and several second silicon alkoxides as previously defined.

More particularly, the method according to the invention comprises the following steps:

a) preparing a microemulsion (M _a ) of the water-in-oil type containing said molecule (s) of interest,

b) adding, to the microemulsion (M _a ) prepared in step (a), a compound for the hydrolysis of a silicon alkoxide,

c) adding, to the microemulsion (M _b ) obtained in step (b), at least one first silicon alkoxide as defined above and at least one second silicon alkoxide having at least one group capable of establishing a non-bonding bond; ionic covalent and / or hydrogen with the molecule of interest as previously defined,

d) adding to the microemulsion (M _c ) obtained in step (c) a solvent for destabilizing said microemulsion, and

e) recovering the silica particles incorporating at least one molecule of interest, precipitated during step (d). Step (a) of the process according to the invention therefore consists in preparing a microemulsion (M _a ) of the type water in oil containing at least one molecule of interest. Any technique making it possible to prepare such a microemulsion can be used in the context of the present invention. Thus, it is possible to:

- either prepare a first solution (Mi) and subsequently incorporate one (or more) molecule (s) of interest to obtain the microemulsion (M _a );

or prepare the microemulsion (M _a ) directly by mixing together the various components and thus one (or more) molecule (s) of interest.

 Advantageously, step (a) of the process according to the invention consists in preparing a first solution (Mi) in which at least one molecule of interest is subsequently incorporated. This solution (Mi) is obtained by mixing together at least one surfactant, optionally at least one co-surfactant and at least one non-polar or weakly polar solvent. The surfactant, the co-surfactant and the non-polar or weakly polar solvent may be mixed at once or added one after the other or in groups. Advantageously, they are added one after the other and, in the following order, surfactant then optionally co-surfactant then non-polar or weakly polar solvent.

Mixing is carried out with stirring using a stirrer, a magnetic bar, an ultrasonic bath or a homogenizer, and may be carried out at a temperature between 10 and 40 ° C, preferably between 15 and 30 ° C and more in particular, at room temperature (ie 23 ° C. ± 5 ° C.) for a period of between 1 min and 1 h, in particular between 10 and 45 min and in particular between 15 and 30 min.

 The surfactant (s) that may be used in the context of the present invention aims to introduce hydrophilic species in a hydrophobic environment and may be chosen from ionic surfactants, nonionic surfactants and mixtures thereof By "mixtures" is meant, in the context of the present invention, a mixture of at least two different ionic surfactants, a mixture of at least two different nonionic surfactants or a mixture of at least one nonionic surfactant. ionic agent and at least one ionic surfactant.

 An ionic surfactant may in particular be in the form of a hydrocarbon chain, charged whose charge is counterbalanced by a counter-ion. As non-limiting examples of ionic surfactants, mention may be made of sodium bis (2-ethylhexyl sulphosuccinate) (AOT), cetyltrimethylammonium bromide (CTAB), cetylpyridinium bromide (CPB) and mixtures thereof.

A nonionic surfactant that may be used in the context of the present invention may be chosen from the group consisting of polyethoxylated alcohols, polyethoxylated phenols, oleates, laurates and their mixtures. As non-limiting examples of commercial nonionic surfactants, there may be mentioned Triton X such as Triton X-100; Brij such as Brij -30; Igepal CO such as Igepal CO-520 or Igepal CO-720; Tween such as Tween 20; Spans such as the Span 85.

 By "co-surfactant" is meant in the context of the present invention an agent capable of facilitating the formation of microemulsions and stabilize them. Advantageously, said co-surfactant is an amphiphilic compound chosen from the group consisting of a sodium alkyl sulphate of 8 to 20 carbon atoms, such as SDS (for "sodium dodecyl sulphate"); an alcohol such as an isomer of propanol, butanol, pentanol and hexanol; a glycol and their mixtures. Advantageously, the co-surfactant, when it is present in the solution (Mi), is ii-hexanol.

 Any non-polar or weakly polar solvent is usable in the context of the present invention. Advantageously, said non-polar or weakly polar solvent is a non-polar or weakly polar organic solvent and, in particular, selected from the group consisting of n-butanol, hexanol, cyclopentane, pentane, cyclohexane, n-hexane cycloheptane, heptane, n-octane, isooctane, hexadecane, petroleum ether, benzene, isobutylbenzene, toluene, xylene, cumene, diethyl ether n-butyl acetate, isopropyl myristate and mixtures thereof. Advantageously, the non-polar or weakly polar solvent used in the context of the present invention is cyclohexane.

In the solution (Mi), the surfactant is present in a proportion of between 1 and 40%, especially between 5 and 30% and, in particular, between 10 and 25% by volume relative to the total volume of said solution. When present in the solution (Mi), the co-surfactant is in a proportion of between 1 and 30%, in particular between 5 and 25% and, in particular, between 10 and 20% by volume relative to total volume of said solution. Thus, the non-polar or weakly polar solvent is present in the solution (Mi) in a proportion of between 40 and 98%, in particular between 50 and 90% and, in particular, between 60 and 80% by volume relative to to the total volume of said solution.

Once the solution (Mi) has been prepared, at least one molecule of interest as defined above is incorporated to form the microemulsion (M _a ) of the water-in-oil type.

The molecule (s) of interest can be added in solid form, in liquid form or in solution in a polar solvent. Whatever the variant used, a polar solvent is added to the microemulsion after incorporation of the molecule (s) of interest in the solution (Mi). Advantageously, the molecule (s) of interest are (are) added to the solution (Mi) in solution in a polar solvent and then the polar solvent, identical or different from the first, is added. More particularly, the two polar solvents used are identical. The addition of the molecule (s) of interest and optionally the polar solvent may be carried out with stirring using a stirrer, a magnetic bar, an ultrasonic bath or a homogenizer. By "polar solvent" is meant in the context of the present invention a solvent selected from the group consisting of water, deionized water, distilled water, acidified or basic, acetic acid, hydroxylated solvents such as methanol and ethanol, low molecular weight liquid glycols such as ethylene glycol, dimethyl sulfoxide (DMSO), acetonitrile, acetone, tetrahydrofuran (THF) and mixtures thereof.

The polar solvent (polar solvent in which the molecule (s) of interest is (are) in solution and / or other polar solvent subsequently added) is present in the microemulsion (M _a ) in a proportion of between 0.25 and 20%, especially between 0.5 and 10% and, in particular, between 1 and 5% by volume relative to the total volume of said microemulsion. The molecule (s) of interest is (are) present in this polar solvent in an amount of between 0.05 and 10%, in particular between 0.1 and 5% and, in particular, between 0.2 and 1.5% by volume relative to the total volume of polar solvent.

Step (b) of the process according to the invention aims to provide for the hydrolysis of the silicon alkoxides by adding to the microemulsion (M _a ) a compound allowing this hydrolysis, the microemulsion (M _b ) thus obtained being a microemulsion water in oil.

By "compound allowing the hydrolysis of a silicon alkoxide" is meant in the context of the present invention a compound selected from the group consisting of ammonia, sodium hydroxide (KOH), lithium hydroxide (LiOH) and sodium hydroxide (NaOH) and, advantageously, a solution of such a compound in a polar solvent, which is identical or different, to the polar solvent used at the time of step (a). The compound for the hydrolysis of a silicon alkoxide is, more particularly, ammonia or a solution of ammonia in a polar solvent. In fact, ammonia acts as a reactant (H2O) and as a catalyst (N¾) for the hydrolysis of a silicon alkoxide.

The compound selected from the group consisting of ammonia, sodium hydroxide (KOH), lithium hydroxide (LiOH) and sodium hydroxide (NaOH), dissolved in the polar solvent, is present in a proportion between 5 and 50%, especially between 10 and 40% and, in particular, between 20 and 30% by volume relative to the total volume of said solution. In addition, said solution is present in a proportion of between 0.05 and 20%, especially between 0.1 and 10% and, in particular, between 0.2 and 5% by volume relative to the total volume of the microemulsion ( M _b ).

Step (b) can be carried out with stirring using a stirrer, a magnetic bar, an ultrasonic bath or a homogenizer, and at a temperature of between 10 and 40.degree. C., advantageously between 15 and 30.degree. more particularly, at room temperature (ie 23 ° C. ± 15 ° C.) for a period of between 5 and 45 minutes, in particular between 10 and 30 minutes and, in particular, for 15 minutes. Step (c) consists in incorporating, into the microemulsion (M _b ) thus obtained, the silicon alkoxides as defined above which will give by sol-gel reaction the silica of the silica particles according to the invention. The incorporation into the microemulsion (M _b ) of the silicon alkoxides to obtain the microemulsion (M _c ) of the water-in-oil type is carried out with stirring using a stirrer, a magnetic bar, an ultrasonic bath or a homogenizer, and can be implemented at a temperature of between 10 and 40 ° C, advantageously between 15 and 30 ° C and, more particularly, at room temperature (ie 23 ° C 15 ° C) for a period of between 6 and 48 h, especially between 12 and 36 h and, in particular, for 24 h.

The first silicon alkoxide (s) and the second silicon alkoxide (s) may be incorporated simultaneously into the microemulsion (M _b ). Alternatively, they can be incorporated successively. In this case, the second silicon alkoxide (s) are advantageously incorporated before the first silicon alkoxide (s).

In the microemulsion (M _c ), the silicon alkoxides ie the first (s) + second (s) alkoxides of silicon are present in a proportion of between 0.05 and 20%, in particular between 0.1 and 10%, and in particular, between 0.5 and 5% by volume relative to the total volume of said microemulsion. Advantageously, the molar ratio of the first (s) alkoxide (s) of silicon / second (s) alkoxide (s) of silicon is between 1 / 0.005 and 1 / 0.5; in particular between 1 / 0.01 and 1 / 0.1; and in particular between 1 / 0.02 and 1 / 0.05.

Step (d) of the process according to the invention aims at precipitating the silica particles by addition of a solvent which does not denature the structure of the nanoparticles but which destabilizes or denatures the microemulsion (M _c ) obtained in step ( vs) .

Advantageously, the solvent used is a polar solvent as defined above. A particular polar solvent to be used in step (d) is chosen from the group consisting of ethanol, acetone and THF. Thus, is added to the microemulsion (M _c ), a volume of solvent identical to or greater than the volume of said microemulsion, in particular greater by a factor of 1.2; in particular, greater by a factor of 1.5; and even greater by a factor of 2 or 3. Any technique making it possible to recover the silica particles incorporating at least one molecule of interest precipitated during step (d) may be implemented during step (e). ) of the process according to the invention. Advantageously, this step (e) implements one or more steps, identical or different, chosen from the centrifugation, sedimentation and washing steps. The washing step (s) is (are) carried out in a polar solvent as defined above. When the recovery step uses several washes, the same polar solvent is used for several even for all washings or several different polar solvents are used at each wash. Regarding one (or more) centrifugation stage (s), it (they) can be implemented by centrifuging the silica particles, in particular in a washing solvent at ambient temperature, at a speed between 4000 and 8000 rpm and, in particular, of the order of 5000 rpm (ie 5000 ± 500 rpm) and this, for a period of between 5 min and 2 h, in particular between 10 min and 1 h and, in particular, during 15 min.

The method according to the present invention may comprise an additional step, following step (e), aimed at removing the molecule (s) of free interest (s) and any trace of surfactant. Advantageously, this step consists in bringing the silica particles recovered following step (e) into contact with a very large volume of water. By "very large volume" is meant a volume greater by a factor of 50, in particular by a factor of 500 and, in particular, by a factor of 1000 to the volume of silica particles recovered after step (e) of the process according to the invention. This step may be a dialysis step, the silica nanoparticles encapsulating one (or more) molecule (s) of interest being separated from the volume by a cellulose membrane, of the Zellu trans type (Roth company). Alternatively, an ultrafiltration step may be provided instead of the dialysis step, via a polyethersulfone membrane. This additional step can, moreover, be implemented with stirring using a stirrer, a magnetic bar, an ultrasonic bath or a homogenizer, at a temperature between 0 and 30 ° C, preferably between 2 and 20 ° C and, more particularly, cold (ie 6 ° C ± 2 ° C) for a period of between 3 h and 36 h, in particular between 6 h and 24 h and, in particular, for 12 h.

For certain applications of the particles according to the invention incorporating at least one molecule of interest, it may be necessary to concentrate them before resuspending them in a suitable liquid or gel. Such a concentration can be obtained, in the case of liquids, by centrifugation. Another known method of biotechnology is to prepare silica particles having magnetic properties. This goal can be achieved through the use of at least one element capable of imparting electromagnetic properties to the particle. This element may be, for example, iron oxide. In this case, the concentration and the recovery of the silica particles according to the invention is done by magnetic field. Therefore, the process of the present invention may have a particular embodiment in which the silica particle incorporating at least one molecule of interest that is prepared is a silica particle comprising at least one element capable of conferring magnetic properties such as a metal component. This form of implementation comprises the addition in the microemulsion (M _a ), in the microemulsion (M _b ) and / or in the microemulsion (M _c ) as previously described of at least one element capable of imparting magnetic properties to the silica particle (iron, gadolinium, nickel, copper, chromium, cobalt, gold, silver, platinum, palladium, or an oxide or hydroxide thereof) in a sufficiently large size small compared to the final size of the desired particle. In this variant, the condensation of the silica with the molecule (s) of interest by the second silicon alkoxide as previously defined is done by incorporating this element and, in the same way, a layer of silica by the first silicon alkoxide as defined above is created on the surface. Advantageously, this element is in the form of a magnetic particle.

The present invention also relates to the microemulsion (M _c ) that can be implemented in the context of the process according to the invention. This microemulsion of water-in-oil type comprises:

 at least one surfactant, especially as defined above,

 optionally at least one co-surfactant, especially as defined above,

 at least one non-polar or weakly polar solvent, especially as defined above,

at least one polar solvent, in particular as previously defined, at least one molecule of interest, in particular as defined above,

 at least one first silicon alkoxide, especially as defined above,

 at least one second silicon alkoxide having at least one group capable of establishing a non-covalent ionic bond and / or hydrogen with the molecule of interest, in particular as defined above,

 at least one compound capable of hydrolyzing said silicon alkoxides, especially as previously defined, and

 optionally an element capable of conferring magnetic properties as previously defined.

 Advantageously, the water-in-oil microemulsion which is the subject of the present invention comprises:

 at least one surfactant in an amount of between 1 and 40%, especially between 5 and 30% and, in particular, between 10 and 25%;

 optionally at least one co-surfactant in an amount of between 1 and 30%, especially between

5 and 25% and in particular between 10 and 20%;

 at least one non-polar or weakly polar solvent in an amount of between 40 and 95%, especially between 50 and 90% and, in particular, between 60 and 80%;

 at least one polar solvent in an amount of between 0.25 and 20%, in particular between 0.5 and

10% and, in particular, between 1 and 5%;

at least one molecule of interest in an amount of between 0.0001 and 2%, in particular between 0, 005 and 0.5% and, in particular, between 0.001 and 0.1%;

 at least one first silicon alkoxide in an amount of between 0.05 and 20%, in particular between 0.1 and 10% and, in particular, between 0.5 and

At least one second silicon alkoxide having at least one group capable of establishing a non-covalent ionic bond and / or hydrogen with the molecule of interest in an amount of between 0.0005 and 0.2%, in particular between 0.001 and 0.1% and, in particular, between 0.005 and 0.05%;

 at least one compound capable of hydrolyzing said silane-based compound in an amount of between 0.01 and 5%, especially between 0.05 and 1% and in particular between 0.1 and 0.5%; and

 optionally an element capable of imparting magnetic properties in an amount of between 0.001 and 5%, especially between 0.005 and 1% and, in particular, between 0.01 and 0.5%;

 the different amounts are expressed in volume relative to the total volume of the microemulsion.

The present invention finally relates to the use of a silica particle according to the invention or capable of being prepared by a process according to the invention in various fields such as sensors, in vivo or in vitro diagnosis, traceability, fight against counterfeiting, preservation and / or transport of molecules of interest. Indeed, as previously explained, the silica particles according to the invention have a number of characteristics listed below:

 protection of the molecule of interest incorporated in the silica particle and in particular in the core of this particle by silica,

maintains in the silica particle of the molecule of interest through the non-covalent bonds between the molecule of interest and the units derived from the 2 ^nd silicon alkoxide;

 porosity of the silica particle which allows the passage of small or large molecules.

 In the application in the sensors, the molecule of interest incorporated in the silica particle according to the invention will be chosen so as to be able to capture a given element.

 In the application in the diagnosis, the silica particle according to the invention via the molecule of interest that it has incorporated can serve as a substrate for biological reactions. Thus, the present invention relates to the silica particle for use as a diagnostic agent.

The experimental part hereinafter shows the use of silica particles incorporating a nucleic acid and in particular an injured DNA to study the activity of repair enzymes or the repairing activity of a given extract such as a cell extract. The silica particles according to the invention can be placed in small volumes of biological extracts containing the enzymes to be assayed. The Silica particles can also be used in cellulo.

 Alternatively, the interior of the silica particle according to the invention can serve as a reactor where the reaction is triggered by the entry of a small substrate which passes through the pores of the silica (hybridization for example in the bloodstream) .

 In the context of anti-counterfeiting and traceability labeling applications, the molecule of interest is advantageously DNA since it makes it possible to encode a large amount of information in a small volume and this information can be specifically amplifiable and decodable. by controlled and known protocols of biotechnology. In fact, DNA marking is already offered on the market by companies. These solutions use the DNA introduced in molecular form in particular liquids.

In the present invention, the experiments described show that the DNA encapsulated in the silica particles remains accessible for, on the one hand, specific amplification (PCR) and, on the other hand, in-situ repair. By the latter method, one can have a decoding of the information by the introduction of a DNA damaged and known in the particle then by a specific repair making it fluorescent and therefore detectable. The fact of having the DNA in a silica particle makes it possible to isolate it sufficiently from the ambient environment in which it is desired to incorporate it to make the marking (for a traceability or anti-counterfeiting application). Encapsulating it in a silica particle also allows access to the functionalization of silica to make these nanotracers compatible with solvents or various materials. For example, it is possible to graft, on the surface of the nanoparticles, hydrophobic functions such as thiol functions, or long carbon or fluorinated chains, and thus be able to disperse these particles in organic solvents. This may for example be used to label polymers or organic liquids.

Other features and advantages of the present invention will become apparent to those skilled in the art on reading the examples hereinafter given by way of nonlimiting illustration, with reference to the appended figures.

BRIEF DESCRIPTION OF THE DRAWINGS

 Figure 1 shows a schematic diagram describing the structure of a silica nanoparticle with the encapsulated plasmid DNA at its core as well as the hydrogen interactions between the di-amino silica network and the phosphate groups of the DNA.

 Figure 2 shows images by transmission electron microscopy of silica nanoparticles according to the invention encapsulating DNA.

FIG. 3 presents the graphs representing the number distribution of silica nanoparticles as a function of size, the silica nanoparticles having been synthesized according to protocol II below: size approximately 40 nm (FIG. 3A) or according to protocol I: size approximately 100 nm (FIG. 3B).

 FIG. 4 shows an image by transmission electron microscopy of silica nanoparticles according to the invention encapsulating polyacrylic acid (protocol IV).

 FIG. 5 shows the analysis by agarose gel electrophoresis of 1% of the non-functionalized silica nanoparticles synthesized with DNA (FIG. 5A) and silica nanoparticles according to the invention, ie functionalized synthesized with DNA (FIG. Figure 5B). FIG. 5A: lanes 1 to 4 (control): molecular weight markers, linearized plasmid (200 ng), supercoiled plasmid (200 ng) and silica nanoparticles, respectively; lane 5: silica nanoparticles prepared in the presence of DNA and then dialysed; lanes 6 to 8: different dilutions of silica nanoparticles prepared in the presence of DNA. Figure 5B: lanes 1 to 3 (control): molecular weight markers, plasmid alone (14 ng, insufficient concentration), silica nanoparticles prepared without DNA; track 4: empty; lane 5: silica nanoparticles encapsulating DNA and lane 6: silica nanoparticles encapsulating DNA-IP.

Figure 6 shows the fluorescent spectroscopy analyzes of IP-labeled DNA in water (Figure 6A), silica nanoparticles encapsulating IP-labeled DNA in water (Figure 6B). ), Cy3 labeled DNA in water (Figure 6C) and silica nanoparticles encapsulating Cy3 labeled DNA (Figure 6D). Figure 7 shows the emission spectra of the nanoparticles analyzed by confocal microscopy obtained after excitation at 488 nm. The nanoparticles studied are silica nanoparticles prepared without DNA (pure silica beads), silica nanoparticles encapsulating DNA (silica / DNA beads), encapsulating DNA-IP (silica / DNA-IP beads). or encapsulating DNA-Cy3 (silica beads / DNA-Cy3) excited at 488 nm.

 FIG. 8 shows the quantization of the fluorescence repair / nick-translation on the 2% agarose gel, using Typhoon 9400 on DNA encapsulated in silica nanoparticles according to the invention encapsulating the DNA with dCTP-Cy3 as fluorescent marker, without "White" enzyme and with "Nick translation" enzyme (FIG. 8A) or with biotin-ddCTP subsequently revealed by streptavidin-Fluoprobes 647, without "White" enzyme and with "Nick" enzyme translation 4 "(Figure 8B).

FIG. 9 shows the quantification of the electrophoretic gel repair tests on silica nanoparticles (Si) and on silica nanoparticles according to the invention encapsulating DNA (Si / DNA), damaged DNA ( If / DNA damage), PAA and DNA (Si / PAA / DNA) or PAA and DNA damage (Si / PAA / DNA damage). DETAILED PRESENTATION OF PARTICULAR EMBODIMENTS

I. Protocol for synthesizing nanoparticles encapsulating DNA.

 1.1. Nanoparticles encapsulating DNA with a size of the order of 100 nm (Protocol I)

 The nanoparticles were prepared using the inverse microemulsion method (water-in-oil) [6].

The microemulsion solution was prepared by mixing the appropriate amounts of surfactant, co-surfactant, organic solvent, water and aqueous ammonia solution, APTMES (3-aminopropyltrimethoxysilane, d = 1.027 M = 179, 29 gmol ^-1 ) and TEOS (tetraethoxysilane, d = 0.934, M = 208, 33 gmol ^-1 ) Ammonia (NH ₄ OH) decomposes as a reactant (H ₂ O). ) and as a catalyst (NH ₃ ) for the hydrolysis of TEOS and APTMES.

 The conventional preparation of an inverse microemulsion for making plasmid-containing silica nanoparticles is described below. This protocol makes it possible to obtain particles of the order of 100 nm (see TEM photos in FIG. 2).

 The procedure involves adding to a 50 mL flask, in the order described, the following chemicals: the triton X100 surfactant (2.1 mL), the n-hexanol co-surfactant (2.05 mL), the cyclohexane organic solvent (9.38 mL). The solution is then stirred at room temperature for 15 minutes.

Then, the plasmid (100 μl at 5 μg / μL in water, 3000 bp), water (200 μL) and ammonia at 33% (125 L, hydrolysis catalyst silicon alkoxides) are added to the solution. The emulsion formed is stirred for 15 minutes.

 The alkoxides of silicon APTMES (1.5 L) and TEOS (123.75 L) are injected into this emulsion. The injection can be done successively starting with the APTMES or simultaneously. In both cases, the results obtained are identical. The reaction is then stirred for 24 h at room temperature.

The emulsion is finally destabilized by the addition of ethanol (45 ml) and the nanoparticles are rinsed three times with ethanol and once with water. Each wash is followed by centrifugation at 5000 rpm for 15 min to sediment the nanoparticles. The silica nanoparticles are vortexed in water (5 mL). They are then dialyzed overnight in 600 ml of H ₂ 0 (MWCO: 4000-6000), which allows the removal of free DNA, and any traces of surfactant. They are then ready to be characterized and used.

1.2. Nanoparticles encapsulating DNA with a size of about 50 nm (Protocol II)

 To obtain 50 nm particles, the protocol is modified by changing the water / surfactant / oily phase ratio.

The procedure involves adding to a 50 mL flask, in order, the following chemicals: Triton surfactant X100 (6.7 mL), co-surfactant n-hexanol (6.6 mL), organic solvent cyclohexane (15 mL). The solution is then stirred at room temperature for 15 minutes. Then, the plasmid (100 μl at 5 μg / μL in water, 3000 bp), water (300 μL) and 33% ammonia (100 L, catalyst for the hydrolysis of the alkoxides of silicon) are added to the solution. The emulsion formed is stirred for 15 minutes.

 The alkoxides of silicon APTMES (1.5 L) and TEOS (98.5 μl) are injected into this emulsion.The reaction is stirred for 24 h at room temperature.

The emulsion is finally destabilized by the addition of ethanol (45 ml) and the nanoparticles are rinsed three times with ethanol and once with water. Each wash is followed by centrifugation at 5000 rpm for 15 min to sediment the nanoparticles. Silica nanoparticles dispersed by vortex in water (5 mL). They are then dialyzed overnight in 600 ml H ₂ 0 (MWCO: 4000-6000), which allows the removal of small residual molecules. They are then ready to be characterized and used.

1.3. Nanoparticles encapsulating DNA with a size of the order of 15 nm (Protocol III)

 To obtain even smaller particles (of the order of 15 nm) it is possible to use another surfactant, according to the protocol below.

 1.3 ml of IGEPAL C0-520 surfactant are mixed with 10 ml of cyclohexane in a flask with magnetic stirring. The mixture is at room temperature and for 30 minutes.

 Then a plasmid solution is introduced

(100 μL at 5 μg / μL in water) followed by a volume of 380 μΐ of water. Then a volume of 100 of ammonia at 33% is added to the emulsion still stirring.

 The solution is stirred for 30 minutes in order to stabilize the system before the successive or simultaneous addition of the silica precursors in the same way as for the other protocols, namely: 1.5 L APTMES and 98.5 μΐ, TEOS. In the case of a successive addition, we first add the APTMES and then the TEOS. The solution is stirred for 24 h.

The emulsion is finally destabilized by the addition of ethanol (45 ml) and the nanoparticles are rinsed three times with ethanol and once with water. Each wash is followed by centrifugation at 5000 rpm for 15 min to sediment the nanoparticles. The silica nanoparticles are vortexed in water (5 mL). They are then dialyzed overnight in 600 ml of H ₂ O (MWCO: 4000-6000), which allows the removal of free DNA and small residual molecules. They are then ready to be characterized and used.

However, in this case, if it is desired to encapsulate DNA, it is necessary to use a smaller plasmid (<3000 bp) or even DNA strands. Indeed, the final size of the nanoparticles also determines the size of the molecules that it is desired to encapsulate. II. Protocol for synthesizing nanoparticles encapsulating polyacrylic acid (Protocol IV)

The nanoparticles were prepared using protocol I. The only difference lies in the step where the polyacrylic acid polymer (100} i ^~ L at 5 g / L in water, a concentration of 0.4 μΜ, MW = 1,250,000 gmol ^-1 ) is introduced into the synthesis in place of the DNA. III. Protocol for synthesizing nanoparticles encapsulating both DNA and polyacrylic acid (Protocol V)

 2.1 ml of Triton X100 surfactant are mixed with 2.05 ml of n-hexanol and 9 ml of cyclohexane in a flask. The mixture is at room temperature and for 15 minutes.

Then 50 μL of polyacrylic acid solution (MW = 1 250 000 gmol ^-1 , C = 0.4 μM) and a plasmid solution (100 L to 5 g / L in water, 3000 bp) are introduced. a volume of 150 μL of water.

Then a volume of 125 L of ammonia 33% is added to the emulsion still stirring. The solution is stirred for 30 minutes in order to stabilize the system before the successive or simultaneous addition of the precursors of silica in the same way as for the other protocols namely: 1.5% APTMES and 123.6%. from TEOS. In the case of a successive addition we start by adding the APTMES then the TEOS. The solution is stirred for 24 h.

The emulsion is finally destabilized by the addition of ethanol (45 mL) and the nanoparticles are rinsed three times with ethanol and once with water. Each wash is followed by centrifugation at 5000 rpm for 15 min to sediment the nanoparticles. The vortex-dispersed silica nanoparticles in water (5 mL) are dialyzed 24 hours. The dialyzed particles are then ready to be characterized and used (M CO: 4000-6000).

 The polyacrylic acid / DNA ratio can be modified to obtain larger objects with a finer silica layer.

IV. Characterization of nanoparticles encapsulating DNA and / or polyacrylic acid

 The results and characterizations presented below were obtained on silica nanoparticles in which plasmids were incorporated by reverse micelle synthesis. This characterization has demonstrated:

 on the one hand, the efficiency of the process of encapsulation of DNA and

on the other hand, the functionality of these objects: that is to say the accessibility of the encapsulated DNA for several types of DNA specific biological reactions, despite the fact that it is encapsulated in the nanoparticles. This is because the shell of the particle is porous silica. IV.1. Morphological characterization

 i. DNA encapsulation

 The MET images of the silica particles with the DNA encapsulated according to the protocol I are shown in FIG. 2. These images show that the particle size is between 50 and 80 nm.

 Moreover, a very clear contrast is visible between the core of the particle and the surface layer. This contrast is due to a difference in electron density which clearly shows that the outer layer of the particles is essentially composed of silica and that the heart is made up mainly of DNA.

 A dynamic light scattering particle measurement (DLS for "Dynamic Light Scattering", Nanosizer from Malvern) confirms the MET observations (Figure 3). ii. Encapsulation of Polyacrylic Acid In order to reinforce this point, a similar experiment was conducted by encapsulating this time a polymer of structure similar to DNA and whose behavior with respect to silicon alkoxides and sol-gel synthesis is similar to DNA (protocol IV). This polymer is polyacrylic acid. The synthesis protocol used is identical to that used in the case of DNA except that the DNA is replaced by polyacrylic acid (see point II).

The MET characterization of the obtained particles shows similar structures with a lesser heart dense than the shell. This characterization thus shows that molecules such as DNA or polyacrylic acid are preferentially found in the core of the particles and that an outer shell of silica is formed (Figure 4).

 In the case of polyacrylic acid, the particle size is much more polydisperse than with DNA, with particles ranging from 40 nm to 150 nm. This is probably due to the disruption of the emulsion by the polyacrylic acid poorly dissolved in the aqueous phase. This dispersion is much less important in the case of encapsulation of the DNA.

IV.2. Characterization of the surface potential

In order to verify that the surface of the nanoparticles is well composed of silica and not of DNA, a measurement of zeta potential was made on beads without DNA and with DNA.

 At pH 7, the potential of the silica particles is -30 mV and that of the nanoparticles encapsulating DNA of -25 mV.

 As the particle surface potential of the two syntheses is almost identical, this measurement shows that the surface consists mainly of silica and that the DNA is confined mainly to the core of the silica particle.

IV.3. Demonstration of the presence of DNA in nanoparticles by electrophoretic method

In order to demonstrate that the previously described method using a silica precursor with amino functions (functionalized silica nanoparticles) allows the stable encapsulation of the DNA in the nanoparticles, a comparative analysis of the agarose gel migration profile of the different preparations is carried out in the presence of the appropriate controls.

Thus, by this method, the DNA trapping capacities of nanoparticles prepared with a silica without amine function (denoted Si) or with amino functions (called "functionalized" denoted SiNH ₂ ) (Protocol I) are compared.

 It is thus possible to differentiate the preparations for which the DNA is trapped in the nanoparticles from those for which the DNA is simply adsorbed on the beads.

The following properties of 1% agarose gel electrophoresis are used: the nanoparticles remain confined in the deposition well, the non-trapped DNA migrates into the gel under the force of the applied electric current. The DNA fragments are separated according to their molecular weight and their bulk (1 kb has a molecular weight of 330,000 g, mol ^-1 ). The negatively charged DNA migrates to the anode as the positive ions of the buffer migrate to the cathode thereby slowing the migration of the DNA by separating these fragments.

Figure 5 shows the electrophoretic migration profile of the two nanoparticle / DNA preparations in the presence of adequate controls. The DNA is revealed by BET. The silica nanoparticles deposited on the gel were synthesized from an emulsion containing the DNA and only TEOS as precursor of silica (denoted Si) (FIG. 5A). The silica beads deposited on the gel were synthesized with DNA, TEOS and a proportion of APTES following the previously described protocol (denoted Si / NH ₂ ) (Protocol I) (FIG. 5B).

 For sample Figure 5A, the plasmids were encapsulated by adding them to the aqueous phase of the micellar synthesis. The agarose gel electrophoretic profile of these nanoparticles was compared to unencapsulated free plasmids (lane 3) and DNA - free nanoparticles (lane 4). Figure 5A shows the silica nanoparticles synthesized with plasmids prior to dialysis (lanes 6, 7 and 8) and after dialysis (lane 5) as well as silica nanoparticles alone (lane 4) and unencapsulated plasmids (lane 3).

 This experience shows that in tracks 6,

7 and 8, the plasmids, which migrated as the unencapsulated control plasmid (lane 3), are free. In addition, after dialysis (lane 5), the plasmids are no longer visible in the gel, so they were removed during dialysis. It is concluded that plasmids are pushed to the surface during nanoparticle synthesis and desorb in dialysis.

The results obtained with the nanoparticles synthesized according to the protocol I from the amino functions of the APTES, are shown in FIG. 5B. The agarose gel shows that the nanoparticles localize with the plasmids revealed by BET (lanes 5 and 6). These plasmids remain in the wells, there is no desorption during gel migration. This experiment tells us that thanks to the functionalization, the plasmids remain attached to the nanoparticles. In addition, there is no electrophoretic separation, the DNA is therefore inside the nanoparticles, trapped by silica and the BET enters the porous silica matrix to intercalate between the base pairs of DNA.

IV.4. Demons ration of the presence of DNA in nanoparticles by confocal microscopy and spectrometry

 Fluorescence analyzes and confocal microscopy were performed to confirm the presence of DNA in the functionalized nanoparticles. To detect the DNA, it was first labeled with different fluorophores such as propidium iodide (IP, by simple incubation between DNA and IP) and Cy3 (by enzymatic labeling by nick - translation). We then encapsulated these labeled plasmids by the functionalised nanoparticle method (Protocol I). The results obtained are described below. i. Fluorescence analysis

The graphs 6A and 6B compare the fluorescence of the IP in water (FIG. 6A) and silica nanoparticles having encapsulated the labeled DNA with the IP intercalator (FIG. 6B). The same excitation and emission curves are observed in both cases. DNA is therefore present in the silica particles.

 Figure 6C shows fluorescence analyzes of Cy3-labeled DNA by nick translation in water compared to that of nanoparticles encapsulating this same Cy3 labeled DNA (Figure 6D). Figure 6D shows the presence of the fluorescence emission peak of Cy3 in the silica nanoparticles. DNA is therefore present in silica nanoparticles. ii. Confocal microscopy analysis

 According to the confocal microscopy images, nanoparticles containing no labeled DNA are not visible (beads + IP alone, beads + unlabeled DNA, beads alone), unlike those prepared with labeled DNA. fluorescent.

Polyvinyl acid (PVA) based films were prepared, and the nanoparticles studied were incorporated into these films in order to study the fluorescence by confocal microscopy. The emission spectra of these nanoparticles are grouped together in FIG. 7. These curves agree with those of FIG. 6, which confirms the presence of fluorophores in the nanoparticles of silicas. V. Demonstration of Enzymatic Accessibility of DNA Encapsulated in the Nanoparticles According to the Invention

 The presence of DNA and its accessibility are evidenced by biological reactions taking place specifically on DNA. This allows the specific labeling of the DNA and therefore its detection.

V.I. Marking and repair of nick-translation-encapsulated DNA

 Previous results demonstrate that synthesized silica nanoparticles and functionalised by reverse micellar pathway are able to retain in their heart plasmids. These plasmids remain accessible to small BET molecules that diffuse through the pores of the silica network.

 The following experiments are intended to demonstrate that the encapsulated DNA is also accessible to large enzyme-like molecules that use DNA as a substrate, in the enzymatic sense of the term.

 Enzymatic labeling was performed by incubating the nanoparticles / DNA (Protocol I) with commercial enzymes and different labeled nucleotides. A control reaction (nanoparticles in the same enzyme-free reaction mixture called "white") is conducted in parallel.

The well-known method of nick-translation biologists has been used via a commercial kit (N5500, Amersham, GE Healthcare) in the presence dCTP-Cy3 or ddCTP-biotin. In the latter case, further incubation in the presence of streptavidin-FluoProbes 647 (Interchim) is necessary to reveal the incorporation of ddCTP-biotin into the DNA.

Experimental protocol :

 The beads (40 μl) are incubated with 21 μl of each of the dATP, dGTP, dTTP of the kit (300 μΜ solutions). 6 μl of dCTP (300 μl), 3 μl of dCTP-Cy3 or 3 μl of ddCTP-biotin (Perkin Elmer) at 1 mM and 30 μl of the enzymatic mixture of the kit (containing a mixture of DNA polymerase I and DNAse I). The reaction is carried out for 4 hours at 15 ° C. and is stopped by adding 6 μl of 0.5 M EDTA. The beads are washed twice with 150 μl of PBS containing 0.2 M NaCl and 0.1% Tween 20. then 2 times with distilled water.

 In the case where the marker used is ddCTP-biotin, 100 μl of this reaction are then incubated with 40 μl of streptavidin-FluoProbes 647 (Interchim) for 15 min at room temperature. The mixture is washed as previously described.

 The nanoparticles are taken up in 100 μl of distilled water.

 An identical quantity (10 μl) of the different nick-translation reactions is deposited in the loading wells of a 2% agarose gel.

In order to determine if a nick-translation labeling has indeed taken place at the DNA level, the fluorescence of the nanoparticles encapsulating DNA is measured, after elimination of the nucleotides not embedded in the DNA, by electrophoresis of the agarose gel reaction mixtures. The labeling solutions are deposited in the loading wells of a 2% agarose gel. During electrophoresis, the nanoparticles remain in the deposition well, while free DNA and negatively charged unincorporated nucleotides migrate into the agarose towards the anode. This is particularly visible on the tracks in which pure dCTP-Cy3 and dCTP-Cy5 have been deposited, the Cy5 being equivalent to FluoProbes 657 in terms of fluorescence spectrum. Quantitation of the fluorescence is performed with a Typhoon 9400 reader (GE Healthcare). This device allows the simultaneous quantification of several fluorophores.

 FIG. 8A shows the quantification of the fluorescence present in the wells corresponding to the ncy-translation test carried out with dCTP-Cy3 as fluorescent marker, without "white" enzyme and with "Nick translation" enzyme.

 FIG. 8B shows the quantification of the fluorescence present in the wells corresponding to the nick-translation test carried out with biotin-ddCTP subsequently revealed by Streptavidin-Fluoprobes 647, without "White" enzyme and with "Nick translation 4" enzyme. ".

The histograms show that the fluorescence is greater in the wells corresponding to the reactions carried out in the presence of enzymes. This means that enzymatic labeling has indeed taken place at the level of the DNA trapped in the nanoparticles. DNA is therefore easily accessible to molecules of the external medium of the nanoparticles.

V.2. DNA repair test encapsulated by excision resynthesis

 During the DNA repair test, the nanoparticles prepared with plasmid DNA or not are incubated with active cell extracts (HeLa nuclear extracts), ATP and fluorophore-labeled nucleotides.

 The cell extract contains, among other things, the enzymes responsible for DNA repair by excision resynthesis (basic excision repair and excision repair of nucleotides), ATP is essential for the catalysis of certain enzymatic reactions and fluorescent nucleotides will be incorporated into the DNA by the polymerases contained in the extracts if the repair takes place.

 This experiment was carried out with the nanoparticles / DNAs prepared with Protocol I, using uninjured DNA as well as damaged DNA.

 This experiment was also performed using nanoparticles / DNA prepared according to protocol V (polyacrylic acid / DNA mixture).

Damaged DNA, i.e., with base lesions, was obtained by UVC irradiation at a dose of 4.5 J / cm ² of the uninjured plasmids. The repair reaction was also performed with the different beads prepared without DNA.

To perform a DNA repair test, the various components are added, in the adequate proportions. In a 50 μΐ final volume tube, the 5 × ATG buffer (200 mM Hepes KOH pH 7.8, 35 mM MgCl ₂ , 2.5 mM DTT, 1.25 μl dATP, 1.25 μM dT P, 1.25 μΜ dGTP, 10 mM EDTA, 17% glycerol, 50 mM phosphocreatine, 250 μg / ml creatine phosphokinase, 0.5 mg / ml BSA), ATP (0.5 μΐ, 100 mM solution), dCTP-Cy5 (0.25 μΐ of a 10 ^{~ 5} M solution), HeLa nuclear extract (1 μΐ to 10 mg / ml, CilBiotech, Belgium), water and nanoparticles dispersed in water. The mixture is incubated for 4 hours at 37.degree. After reaction, the nanoparticles are centrifuged and washed twice with 150 μl of PBS containing 0.2 M NaCl and 0.1% Tween 20, once with ethanol and then twice with distilled water. The nanoparticles are then dispersed in 100 μl of water.

 An identical quantity (10 μl) of the different repair reactions is deposited in the loading wells of a 2% agarose gel.

 The fluorescence measurement is carried out after elimination by electrophoresis on agarose gel nucleotides not incorporated in the DNA. During electrophoresis, the beads remain in the deposition well, while free DNA and unincorporated nucleotides migrate into the agarose to the anode.

 Quantitation of the fluorescence in each well is performed with a Typhoon 9400 reader (GE Healthcare). The values are reported in the histogram Figure 9.

On this histogram, the amount of fluorescence is clearly higher when the encapsulated DNA has been previously damaged, than with a DNA without lesion, or no DNA at all. The repair reactions therefore took place at the level of the nanoparticles encapsulating the DNA. 1 / Encapsulated DNA is therefore well accessible to enzymes contained in the external medium and the reaction that takes place is very specific.

The encapsulated DNA can therefore serve as a substrate for the assay of enzyme activities present in the external medium.

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Claims

1) porous silica nanoparticle, incorporating at least one molecule of interest, characterized in that the silica network inside said nanoparticle is functionalized by at least one group capable of establishing a non-covalent ionic bond and / or hydrogen with the molecule of interest, whereby the molecule (s) of interest is (are) bound to the silica network solely by non-covalent bonds.

2) silica nanoparticle according to claim 1, characterized in that the groups capable of establishing a non-covalent ionic bond and / or hydrogen with the molecule of interest functionalizing said nanoparticle are distributed in the latter in the form of a decreasing gradient of the center of the nanoparticle towards the outside of the nanoparticle, no group being present on the surface of the nanoparticle.

3) silica nanoparticle according to claim 1 or 2, characterized in that the silica nanoparticle is mesoporous with an open porosity.

4) silica nanoparticle according to claim 1 to 3, characterized in that said molecule of interest is selected from the group consisting of an enzyme, a protein, an oligopeptide, a peptide, an antigen, an antibody, a nucleic acid, a polymer and a carbohydrate.

5) silica nanoparticle according to any one of the preceding claims, characterized in that said group capable of establishing a non-covalent ionic bond and / or hydrogen is selected from the group consisting of -NH ₂ , -NHR ₁₂ with R ₁₂ representing an alkyl radical of 1 to 6 carbon atoms, -NH ₃ ^{+, +} -N¾Ri3 with R13 representing an alkyl radical of 1 to 6 carbon atoms, -COOH, -COO ^", C (0) NH, -C (O ), -SH and -OH.

6) silica nanoparticle according to any one of the preceding claims, characterized in that it comprises at least one element capable of conferring magnetic properties.

7) Process for preparing a silica nanoparticle incorporating at least one molecule of interest according to any one of claims 1 to 6, characterized in that it comprises the preparation, in the presence of said molecule of interest, from minus one silica particle per inverse emulsion from:

at least one first silicon alkoxide of the formulas Si (ORi) ₄ , R ₂ Si (OR ₃ ) 3 or R ₄ R ₅ Si (OR ₆ ) 2 in which R 1, R ₃ and R 6, which may be identical or different, represent an alkyl radical of 1 to 6 carbon atoms and R ₂ , R 4 and R ₅ , identical or different, represent a hydrogen, an alkyl radical of 1 to 6 carbon atoms or an alkenyl radical of 1 to 6 carbon atoms, and

 at least one second silicon alkoxide having at least one group capable of establishing a non-covalent ionic bond and / or hydrogen with the molecule of interest.

8) Process according to claim 7, characterized in that the radicals] ¾, ¾ and R5 of said first silicon alkoxide, identical or different, are selected from the group consisting of hydrogen, methyl, ethyl, vinyl and propyl.

9) Process according to claim 7 or 8, characterized in that said first silicon alkoxide is selected from the group consisting of tetramethoxysilane, tetraethoxysilane, tetrapropoxysilane, tetrabutoxysilane, trimethoxysilane, methyltrimethoxysilane, ethyltrimethoxysilane, propyltrimethoxysilane, vinyltrimethoxysilane, triethoxysilane, methyltriethoxysilane, ethyltriethoxysilane, propyltriethoxysilane, vinyltriethoxysilane, and mixtures thereof.

10) A method according to any one of claims 7 to 9, characterized in that said second silicon alkoxide is R7Si formulas (ORs) 3 or R9R ₁ 0S1 (ORn) ₂ in which Rs and R, identical or different, represent an alkyl radical of 1 to 6 carbon atoms and R ₇ , R ₈ and R ₁₀ , which are identical or different, represent an alkyl radical of 1 to 8 carbon atoms, a heteroalkyl radical of 1 to 10 carbon atoms, an alkylaryl radical of 1 to 12 carbon atoms or an alkenyl radical of 1 to 8 carbon atoms,

 the radical R and at least one of the radicals R9 and Rio being substituted by at least one group capable of establishing a non-covalent ionic bond and / or hydrogen with the molecule of interest.

11) Process according to any one of claims 7 to 10, characterized in that said second silicon alkoxide used in the context of the present invention is selected from the group consisting of N-trimethoxysilylpropyl-N, N, N chloride trimethylammonium, 1-aminoethylaminomethyl) phenethyltrimethoxysilane, N- (6-aminohexyl) aminopropyltrimethoxysilane, 3-aminopropyl-methyl-diethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3- (2-aminoethylamino) ) propyl-trimethoxysilane, (3-mercaptopropyl) trimethoxysilane, (3-mercaptopropyl) triethoxysilane and mixtures thereof. 12) Method according to any one of claims 7 to 11, characterized in that said method comprises the following steps:

a) preparing a microemulsion (M _a ) of the water-in-oil type containing said molecule (s) of interest, b) adding, to the microemulsion (M _a ) prepared in step (a), a compound for the hydrolysis of a silicon alkoxide,

c) adding, to the microemulsion (M _b ) obtained in step (b), said at least one first silicon alkoxide and said at least one second silicon alkoxide having at least one group capable of establishing a non-ionic covalent bond and / or hydrogen with the molecule of interest,

d) adding to the microemulsion (M _c ) obtained in step (c) a solvent for destabilizing said microemulsion, and

 e) recovering silica nanoparticles incorporating at least one molecule of interest, precipitated during step (d).

13) Process according to claim 12, characterized in that said step (a) of the process consists in preparing a first solution (Mi) in which at least one molecule of interest is subsequently incorporated, said solution (Mi) being obtained by mixing together at least one surfactant, optionally at least one co-surfactant and at least one non-polar or weakly polar solvent.

14) Process according to claim 12 or 13, characterized in that at least one element capable of imparting magnetic properties to said silica particle is added in said microemulsion (M _a ), said microemulsion (M _b ) and / or in said microemulsion (M _c ). 15) microemulsion water-in-oil type (M _c ) may be implemented in the context of a method according to any one of claims 12 to 14, characterized in that it comprises:

 at least one surfactant,

 optionally at least one co-surfactant, at least one non-polar or weakly polar solvent,

 at least one polar solvent,

 at least one molecule of interest at least one first silicon alkoxide, at least one second silicon alkoxide having at least one group capable of establishing a non-covalent ionic bond and / or hydrogen with the molecule of interest,

 at least one compound capable of hydrolyzing said silicon alkoxides, and

 optionally an element capable of imparting magnetic properties.

16) Microemulsion according to claim 15, characterized in that it comprises:

 at least one surfactant in an amount of between 1 and 40%, especially between 5 and 30% and, in particular, between 10 and 25%;

 optionally at least one co-surfactant in an amount of between 1 and 30%, especially between 5 and 25% and, in particular, between 10 and 20%;

at least one non-polar or slightly polar solvent in an amount of between 40 and 95%, especially between 50 and 90% and, in particular, between 60 and 80%;

 at least one polar solvent in an amount of between 0.25 and 20%, especially between 0.5 and 10% and, in particular, between 1 and 5%;

 at least one molecule of interest in an amount of between 0.0001 and 2%, in particular between 0.005 and 0.5% and, in particular, between 0.001 and 0.1%;

- at least one first silicon alkoxide in an amount between 0.05 and 20%, especially between 0.1 and 10% and in particular between 0.5 and _f o

 at least one second silicon alkoxide having at least one group capable of establishing a non-covalent ionic and / or hydrogen bond with the molecule of interest in an amount of between 0.0005 and 0.2%, in particular between 0.001 and 0, 1% and, in particular, between 0.005 and 0.05%;

 at least one compound capable of hydrolyzing said silane compound in an amount of between 0.01 and 5%, in particular between 0.05 and

1% and in particular between 0.1 and 0.5%; and

 optionally an element capable of conferring magnetic properties in an amount of between 0.001 and 5%, especially between 0.005 and 1% and, in particular, between 0.01 and 0.5%;

the different amounts are expressed in volume relative to the total volume of the microemulsion. 17) Use of a silica nanoparticle according to any one of claims 1 to 6 or capable of being prepared by a method according to any one of claims 7 to 14, in sensors, in vitro diagnosis, traceability , fight against counterfeiting, preservation and / or transport of molecules of interest.

18) silica nanoparticle according to any one of claims 1 to 6 or capable of being prepared by a method according to any one of claims 7 to 14 for use as a diagnostic agent.