EP2242486A1

EP2242486A1 - Encapsulation of vitamin c into water soluble dendrimers

Info

Publication number: EP2242486A1
Application number: EP09720309A
Authority: EP
Inventors: Didier Astruc; Jaime Ruiz Aranzaes; Elodie Boisselier
Original assignee: Centre National de la Recherche Scientifique CNRS
Current assignee: Centre National de la Recherche Scientifique CNRS
Priority date: 2008-01-21
Filing date: 2009-01-21
Publication date: 2010-10-27
Also published as: WO2009112682A1; FR2926548A1; US20110021626A1; FR2926548B1

Abstract

The invention relates to a conjugated dendrimer that comprises at least one water-soluble dendrimer and at least one vitamin C molecule. The conjugated dendrimer can be used e.g. for preparing cosmetic or pharmaceutical compositions. The dendrimers have a good vitamin C load capacity and are biocompatible.

Description

ENCAPSULATION OF VITAMIN C IN DENDRIMERS

SOLUBLE IN WATER

DESCRIPTION

Technical area

The present invention relates to the encapsulation of vitamin C using water-soluble dendrimers, and, in particular, to its method of preparation.

[2] The use of this dendrimer encapsulating vitamin C concerns for example the fields of pharmacy, cosmetics, organic chemistry, and that of green chemistry. [3] In the description below, the references in parentheses (X) refer to the list of references presented after the examples.

State of the art

[4] As the name suggests, a dendrimer is a molecule whose architecture resembles that of the branches of a tree. It is indeed a macromolecule of three-dimensional structure, related to a hyperbranched polymer, where the connected monomers are associated in a tree process around a central multivalent heart. [5] Dendrimers generally take a very regular or spherical globular form, highly branched and multifunctionalized. They consist of three specific regions: a multivalent central core, a defined number (constituting the multivalence) of intermediate dendritic branches connected to the multivalent central heart where each dendritic branch consists of a number of branching generation, and the periphery consisting of a multitude of functional end groups.

[6] These molecules have both internal cavities and a large number of terminal groups at the periphery, easily accessible, which can be responsible for a variety of properties and reactivities.

[7] For example, the document by Tomalia et al.

Macromolecules (1986) 19, page 2466 (1) which describes the construction of the first water-soluble dendrimers. [8] Dendrimers are built step by step using a succession of sequences, each resulting in a new generation. Structural control is critical to the specific properties of these macromolecules.

[9] Dendrimers generally take a very regular or spherical globular form, highly branched and multifunctionalized. In addition, dendrimers are generally monodisperse, unlike hyperbranched polymers. Obtaining these regular structures that are the dendrimers requires synthetic methods other than ordinary polymerizations. Indeed, the synthesis of dendrimers is difficult because it requires many steps to protect the active location, which is an obstacle to the synthesis of significant amounts. In addition, obtaining dendrimers with defined and / or specifically functionalized multiple trees is a real challenge for chemists because it requires quantitative and clean reactions to avoid mixing on the branches. By way of comparison, the hyperbranched polymers, whose molecular architecture is irregular, are obtained by polymerization of multifunctional branched monomers in a disordered and unrestricted process.

[10] Thus, these macromolecules are adjustable: their structure, their flexibility, their porosity and their morphology can be optimized in order to obtain the desired properties. The control of the architecture of the dendrimers and the chemical nature of the terminal branches which can thus perform various functions arouse an increasing interest for many very promising applications, especially in the field of medicine as described for example in the article by Astruc, CR. Acad. ScL (1996), 322, Series 2b, pages 757-766 (2), but also from the food industry, aromas, paints, catalysis, decontamination, gene therapy, photodynamic therapy, biological sensors, nanoelectronics, etc.

[11] On the other hand, the vectorization of active molecules is today a major stake in the pharmaceutical and cosmetic fields, as described for example in the publication by Vandamme et al., J. Control. Release (2005), 102 (1), pages 23-38 (3). It corresponds to the transport of active molecules to a site of action which allows to modulate the pharmacokinetics and biodistribution of these molecules by promoting their presence at the sites of action while limiting their toxicity or impact on healthy tissue . In addition, the vectorization allows a protection of the active molecules vis-à-vis the medium in which they are introduced avoiding for example their metabolism or degradation, as described for example in the publication of Zhuo et al. J. Control. Release (1999) 57, p. 249 (4). [12] Vitamins are of great interest, especially for manufacturers in the pharmaceutical and cosmetic fields. Indeed, they intervene in many biological and physiological processes and are essential for the organism. For example, vitamin C, or ascorbic acid (AA) has many properties. Known for her toning and anti-fatigue actions, she works for example in the synthesis of red blood cells and contributes to the immune system. Thus, it participates in the defenses of the body and thus plays a role in the fight against infections. Vitamin C also promotes the absorption of iron, which helps prevent the onset of anemia. It is also required in the synthesis of collagen in the skin and is essential for healing wounds. It is also a powerful anti-oxidant and antiradical that protects the body from free radicals, prevent oxidative degradation and aging. Thus, these applications, in particular cosmetics, are numerous, also stimulating cellular metabolism and having a photoprotective effect. [13] However, some vitamins, including vitamin C, can not be synthesized by the human body, hence the need for an exogenous supply.

[14] In addition, vitamins are fragile compounds, easily degraded by light, heat, oxygen, etc. In particular, vitamin C, the most fragile of all vitamins, is unstable in solution and is quickly eliminated by the body. Existing pharmaceutical and cosmetic products therefore have a limited time of use. To overcome this problem of instability, they use a very different formulation that does not allow a modulated release. In addition, this molecule is not currently vectorized, its dispersion is uncontrolled and nonspecific.

[15] There is therefore a real need for new compounds to vectorize and control the release of active molecules, in particular cosmetic active ingredients and especially vitamins, for example vitamin C. [16] In addition, there exists a real need to have biocompatible compounds, low toxicity, defined architecture, size and controlled form, high load capacity, specific functionalization to optimize the encapsulation of vitamin C.

[17] In addition, there is a real need for a process for encapsulating vitamin C with good yields, preserving the environment and reducing the cost of obtaining these compounds.

Description of the invention

[18] The present invention is specifically intended to meet these needs and disadvantages of the prior art by providing a conjugated dendrimer comprising at least one water-soluble dendrimer encapsulating at least one molecule of vitamin C. [19] For the purposes of the present invention, the term "conjugated dendrimer" means the combination of at least one water-soluble dendrimer with at least one vitamin C molecule. In addition, a vitamin C molecule may be associated with one or more dendrimers which may be identical or different, for example if the PEG chains of different dendrimers participate in the stabilization of vitamin C molecules. [20] The term "water-soluble dendrimer" of the present invention, a dendrimer which can be dissolved in water, that is to say whose aqueous solution is clear. In particular, the NMR spectrum of this solution makes it possible to visualize all the groups present in the dendrimer. It may be, for example, dendrimers having on their surface hydrophilic and / or little hydrophobic functions, that is to say polar functions and / or functions capable of creating hydrogen bonds with the water molecules. It may be for example dendrimers having on their surface amino functions (-NH 2, -NH- or -N-), ammonium (-NH ₄ ⁺ ), cyano (-CN), amido (-C (= O ) -NH ₂ or -C (= O) -NH-), hydroxyl (-OH), alkoxides (- O ^" ), carboxyl (-C (= O) -OH), carboxylate (-C (= O) - O ^~ ), carbonyl (-C (= O) H or -C (= O) ~), oxy (-O-), ester (-C (= O) -O-), preferably amino, ammonium, amido and oxy. [21] For the purposes of the present invention, the term "encapsulate" is understood to mean surrounding a molecule, for example in the form of a capsule or shell, making it possible to isolate it, stabilize it and / or This encapsulation can be done in the heart but also on the periphery of the dendrimer where the vitamin C molecules are turned towards the interior of the dendrimer and are thus stabilized and protected.

[22] "Association" in the sense of the present invention, the assembly of two or more different molecules, for example by supramolecular bonds. The vitamin C molecules may for example be associated inside the dendrimer or on the surface thereof. For the purposes of the present invention, the term "supramolecular bond" means the assembly of several distinct molecules by hydrogen, ionic, coordination and / or hydrophobic interaction bonds. [24] Surprisingly, the inventors have demonstrated that the vitamin C molecules were encapsulated by the dendrimers of the present invention and did not remain in solution. In other words, the vitamin C molecules associate with either the core or the periphery of the dendrimers of the present invention. The dendrimers of the present invention thus make it possible to stabilize the vitamin C molecules and / or to protect them from the external environment. Indeed, the vitamin C molecules are incorporated into a "capsule" protected by a hydrophilic crown. [25] Moreover, the dendrimers have the advantage of being unique molecules (that is to say of polydispersity equal to one), perfectly defined, with exact chemical formulas, and which can be characterized very precisely ( for example by nuclear magnetic resonance (NMR) proton and / or carbon techniques, microanalysis, infrared spectrometry, or mass spectrometry). Dendrimers are therefore suitable molecules for biological applications that require a high purity and a great knowledge of the molecules introduced in a biological medium, for example their mode of assembly, operation, their stability or the way in which they could be metabolized. Indeed, biological applications are much more difficult to achieve in the case of polydispersed or polymolecular molecules such as hyperbranched polymers. In particular, the hyperbranched polymers comprise a mixture of hyperbranched molecules that do not have the same molecular weight and will not stabilize an identical number of biological molecules for each of them, which generates much more uncertainty and difficulties for them. adapt to biological use.

[26] In addition, the water-soluble dendrimers of the present invention appear to be suitable carriers for the transport of vitamin C molecules, i.e., ascorbic acid. Indeed, the controlled multivalence of the dendrimers of the present invention can be used to attach one or more substances and / or targeting groups and solubilizing at the periphery. This attachment or association can be achieved by supramolecular bonds that allow the maintenance of vitamin C molecules in the heart and periphery of the dendrimers. [27] The dendrimers of the present invention can behave like vectors of vitamin C. The vectorization aims at modifying the stability and the pharmacokinetic properties of the transported active principles such as the crossing of anatomical, physiological barriers, their targeting. The particular characteristics of dendrimers, and their globular form, make these new molecular architectures ideal carriers of charged molecules. Indeed, these molecules can be used in a large number of applications including the control of loosening of pharmaceutical or cosmetic products.

[28] According to a particular embodiment of the invention, the water-soluble dendrimer may have a symmetrical radial structure.

[29] Indeed, the inventors have made "perfect" dendrimers.

For the purposes of the present invention, the term "perfect" dendrimer means a dendrimer of symmetrical structure that has no defect, with a total and uniform functionalization of the branches. The high symmetry of the dendrimer architecture has been verified by different characterization methods (NMR, mass, etc.). These data are exposed in the "Examples" section.

[30] Dendrimers with a symmetrical structure have the advantage of being perfectly controlled and characterized, with reproducible results, which is essential for transposition at the industrial level. However "imperfect" dendrimers may also encapsulate vitamin C molecules. The term "imperfect" dendrimer for the purpose of the present invention means a dendrimer which does not fall within the definition of the so-called "perfect" dendrimer. [31] Furthermore, according to the invention, the water-soluble dendrimer may consist of g generations, g being an integer ranging from 0 to 10, preferably from 0 to 4, in order to respect a permissible size in the body. For the purposes of the present invention, "generation" is understood to mean a repetition of branching units or monomers organized in a layer around the central core. A generation is counted from each division of a branch into at least two branches. The generation number g of the dendrimer corresponds to the number of concentric layers of monomer, but can also correspond to the number of sequences necessary for the synthesis of the dendrimer.

[33] The generation number g of the dendrimer induces a precise size of the dendrimer and also plays an important role in the conformation of the dendrimer.

[34] Advantageously, the water-soluble dendrimer can have a globular structure. It may be for example a lobe structure, an elliptical structure, a spherical structure, perfect or not perfect, preferably a perfect spherical structure.

[35] Indeed, the dendrimers have a dendritic topology that is to say an architecture built according to a tree process around a central core multifunctional, analogically to a neural network dendritic. The connected units are repeated monomers organized in layers, also called generations. According to the dendrimers, beyond a certain number of generations, they can take a globular form if the molecular segments are flexible. [36] The dendrimers of globular structure have the advantage of being able to accommodate molecules within their cavities with a larger capacity. Indeed, the dendritic topology imposes dynamic cavities absent in a linear polymer, for example, whose topology is different. The term "cavities" within the meaning of the present invention means the spaces present between the branches of the dendrimers. Since a dendrimer is not fixed in solution, the cavities can be dynamic because of movement of the branches within the solution, guided by the movement

Brownian.

[37] By way of example, the following comparative Table 1 details the different properties of a linear polymer and a dendrimer. Thus, depending on the desired criteria, the dendrimer may be preferred to obtain the various properties below which differ from those of a linear polymer.

Table 1 [38] In particular, the main advantage of a globular dendrimer over a linear polymer is the size of its internal cavities which can be optimized to accommodate large molecules. Indeed, in the case of the dendrimer, the cavities can be large and organized so as to allow greater capacity of reception or encapsulation. On the other hand, in a linear polymer folded into a spherical ball, the structure is random and the cavities are not optimized.

[39] According to another particular embodiment of the invention, the water-soluble dendrimer may have a diameter of less than 200 A, preferably 10 A to 50 A. This diameter depends on the generation of the dendrimer.

[40] According to the invention, the water-soluble dendrimer may have a molecular weight ranging from 20 to 50,000 g / mol, for example from 500 to 15,000 g / mol, for example from 773 to 7168 g / mol . [41] The water-soluble dendrimer of the present invention may be, for example, a dendrimer of the following formula (I):

Formula (I) in which:

(a) A is the central heart of the dendrimer, of multivalence k, where: k is an integer ranging from 2 to 9; A is a radical chosen from the group comprising a C 1 to C ₂₂ heteroalkyl, a phenyl optionally substituted by one or more C 1 to C ₆ heteroalkyl groups, the radical A possibly comprising one or more carbon substituted by an oxo group ( = O); (b) Mi is a monomer of generation i, wherein: i is an integer from 0 to g, where g is the generation number of the dendrimer; when i = 0, Mi is non-existent, the terminal branch BT is then directly connected to the central core A; when i> 0, Mi is a C 1 -C 22 heteroalkyl radical, in which one or more carbon may optionally be substituted by an oxo (= O) group, for example Mi may be a radical - (CH ₂ ) _a -N * or a radical - (CH ₂ ) _a -C (= O) -NH- (CH 2) _b -N * with a and b are integers from 1 to 6 and the symbol * denotes the point of attachment of the radical Mi au with the higher generation monomer;

(c) BT is the terminal branch, and p is the number of terminal units from the lower generation monomer where: p is an integer from 1 to 3, preferably p is 2 or 3, preferably p is 3; BT is a radical selected from the group consisting of hydrogen, -NH ₂ group, an ammonium group, an -OH group, a heteroalkyl -C 22 -alkyl, phenyl substituted by one or more heteroalkyl groups to C _Θ O, the radical BT possibly comprising one or more carbon substituted with an oxo group (= O); for example, BT may be a radical -C (= O) -CH ₂ -O- (CH ₂ ) ₂ -O- (CH ₃ ) or a radical -C (= O) -Ph ((OC ₂ H ₄ ) 3-OCH3) 3;

For the purposes of the present invention, the term "multivalence" refers to the number of dendritic branches connected to the central core of the dendrimer. For the purposes of the present invention, the term "dendritic branch" is intended to mean a branched branch. In particular, it may consist of a sequence of monomers Mi, identical or different, where each branch between the monomers is divided into two or more branches. For example, in formula (I), the dendritic branch can be: [(M1) - (M2) ... 2 - (Mi) ₂ ^A (M) H (OB].

For the purpose of the present invention, the term "terminal branch" is intended to mean that part of the dendritic branch constituted by the terminal unit located at the periphery of the dendrimer. It may be for example a last-generation monomer g of the dendrimer, a hydrophilic function, a dendron, etc. For example, in formula (I), the terminal branch corresponds to: (BT). [45] For the purposes of the present invention, the term "intermediate dendritic branch" means that part of the dendritic branch connecting the multivalent central core A to the terminal branch BT. For example, in formula (I), the intermediate dendritic branch corresponds to: [(M1) - (M2) ₂ - ... - ]. For the purposes of the present invention, the term "dendron" is intended to mean a branched (for example non-terminal or internal) dendritic branch, for example, comprising 2 or more branches. For example, the dendron may be a hyperbranched dendritic branch, that is to say comprising 3 or more ramifications. As dendron, mention may for example be made of pentafluorophenyl tris 3,4,5-tri (triethyleneoxy) benzoate (or PentaFluoroPhenyl tris 3,4,5-Tri (TriEthyleneGlycol) benzoate or PFPTTEG). [47] The term "alkyl" in the sense of the present invention, a linear radical, branched or cyclic, saturated or unsaturated, optionally substituted, comprising 1 to 60 carbon atoms, for example 1 to 57 carbon atoms, by Example 1 to 28 carbon atoms, for example 1 to 22 carbon atoms, for example 1 to 6 carbon atoms. [48] The term "alkene" in the sense of the present invention, an alkyl radical, as defined above, having at least one carbon-carbon double bond.

[49] The term "alkyne" in the sense of the present invention, an alkyl radical, as defined above, having at least one carbon-carbon triple bond. For the purposes of the present invention, the term "aryl" means an aromatic system comprising at least one ring satisfying the Hekel aromaticity rule. Said aryl is optionally substituted, may be mono or polycyclic and may comprise from 6 to 10 carbon atoms. [51] The term "heteroalkyl" in the sense of the present invention, an alkyl radical as defined above, said alkyl system comprising at least one heteroatom, in particular selected from the group consisting of sulfur, oxygen, nitrogen, silicon.

[52] According to the invention, the water-soluble dendrimer may have a hydrophobic central core and a hydrophilic crown. It is understood that the hydrophobic core does not necessarily include only hydrophobic groups. In particular, the hydrophobic core may contain heteroatoms (for example N, O, S and / or Si). In this sense, "hydrophobic core" means a core with a hydrophobic nature relative to the periphery of the dendrimer, which is hydrophilic. This hydrophobic core / hydrophilic crown system makes it possible, in particular, to maintain the vitamin C molecules in the heart of the dendrimer, thus avoiding their contact. with the external environment (or the biological environment) and therefore premature oxidation of these molecules, while allowing the release of active molecules of vitamin C, especially in a biological medium. ^;

[53] According to one particular embodiment of the invention, the water-soluble dendrimer may have intermediate dendritic branches comprising heteroatoms, for example branches comprising amino groups (-NH- or -N-), amido (-C (= O) -NH-), oxy (-O-) or ester (-C (= O) -O-), preferably amino, for example secondary or tertiary amine. These groups being located inside the dendrimer, this has the advantage of allowing a better encapsulation of vitamin C molecules inside the dendrimer, for example by hydrogen, ionic and / or coordination, and ^; to avoid their contact with the external environment. In addition, this also makes it possible to increase the number of encapsulated vitamin C molecules. [54] According to a particular embodiment of the invention, the water-soluble dendrimer may have at its periphery mainly free primary amine functions or ammonium functions. [55] Indeed, the dendrimers comprising free primary amines or peripheral ammonium functions have the advantage of being able to react either supramolecularly with ascorbic acid (for example by forming ammonium ascorbates), as demonstrated in FIG. the examples part, or covalently with other chemical groups in order to modify the periphery of the dendrimer and therefore its properties. [56] For example, they may be dendrimers comprising a DiAminoButane (DAB) core and poly (propylene) imine (PPI) branches, such as, for example, the dendrimers described in FIGS. 1 to 4, or poly dendrimers ( amido) amines (PAMAM), for example the dendrimers described in FIGS. 5 to 9. [57] Thus, according to one particular embodiment of the invention, the water-soluble dendrimer may have one of the structures following: DAB G2 (Figure 1), DAB G3 (Figure 2), DAB G4 (Figure 3), DAB G5 (Figure 4), PAMAM GO (Figure 5), PAMAM G1 (Figure 6), PAMAM G2 (Figure 7), PAMAM G3 (Figure 8), PAMAM G4 (Figure 9), etc. [58] Some of these dendrimers are commercially available, eg DAB dendrimers G2, G3 DAB, DAB G5 PAMAM G1 and G4 PAMAM are marketed by the company Sigma-Aldrich (Sigma-Aldrich Chemie GmbH, L ¹ Isie Abeau Chesnes , 38297 Saint-Quentin Fallavier, France), respectively under the references No. 679895 (DAB G2), No. 469076 (DAB G3), No. 469092 (DAB G5), No. 597414 (PAMAM G1) and No. 597856 ( PAMAM G4). [59] Depending on the choice of dendrimers used, it is possible to promote the combination of vitamin C molecules preferentially at the periphery and then at the heart of the dendrimers (as for example in the case of PAMAM G4) or conversely to favor first the association with heart.

[60] According to a particular embodiment of the invention, the water-soluble dendrimer may be functionalized at its periphery with at least one organic surface agent.

[61] The term "surfactant" according to the invention a molecule present on the surface of the dendrimer for modulating its surface properties, for example: to modify its solubility to modulate its toxicity and its biodistribution, for example to avoid its recognition by the reticuloendothelial system ("stealth"), and / or to give it interesting bioadhesion properties when administered orally, ocularly, nasally, and / or to allow specific targeting of certain organs / fabrics, etc.

[62] According to a particular embodiment of the invention, the organic surface agent may be selected from the group consisting of: a pharmaceutical molecule, a targeting molecule or a solubilizing group. [63] According to another particular embodiment of the invention, the organic surface agent may be a solubilizing group with polyethylene glycol chains.

[64] Indeed, the functionalization of these dendrimers with different organic groups containing polyethylene glycol chains makes it possible to improve the solubility in water of these macromolecules as described in the document by Liu et al. J. Polym. Sci. : part A: Polym. Chem. (1999), page 3492 (5), as well as biocompatibility. Surprisingly, in addition to these important characteristics mentioned above for a product intended for biological use, dendrimers functionalized with polyethylene glycol chains have proved to be better molecular vectors by their encapsulation efficiency. In addition, these molecular vectors can be modified as desired in order to optimize the encapsulation of vitamin C molecules. Indeed, a dendron containing polyethylene glycol chains not only makes it possible to improve the encapsulation rate of vitamin C molecules. but also to promote the encapsulation inward of the dendrimer (for better protection), for example by making supramolecular bonds with the vitamin C molecules, despite the steric hindrance that may occur. This property was unexpected, and was highlighted by the inventors. As an example, the DAB G3 dendrimer functionalized by the PFPTTEG dendron can fix more than 150 molecules of vitamin C. In addition, this dendrimer (with a hyperramified dendron thus having a high steric hindrance) makes it possible to fix many more molecules. of vitamin C than the DAB G3 dendrimer functionalized by the MEAC ligand. Other examples of encapsulation yields of dendrimers according to the invention are given in the Examples section.

[65] According to a more particular embodiment of the invention, the organic surface agent may be a solubilizing group corresponding to one of the following structures (II) or (III):

Structure (II)

Structure (III) wherein: n, n, r ₂ and r ₃ are independently an integer of 1 to 500, and

R, Ri, R ₂ and R ₃ independently represent an alkyl to C ₆ alkene C2 -C ₆ alkyne C 2 -C _6, or a C ₆ to C 10 optionally substituted.

[66] According to a more particular embodiment of the invention, the solubilizing group may respond to one of the following structures (IV) or (V) in which:

Structure (IV)

wherein n, n, r, and 113 are 1 or 3, preferably n is 1 and n, n ₂ and n ₃ are 3;

[67] For example, the water-soluble dendrimer according to the invention can be a DAB-dendrimer or a PAMAM-dendrimer functionalized with dendron pentafluorophenyl tris 3,4,5-tri (triethyleneoxy) benzoate (or PentaFluoroPhenyl tris 3 , 4,5-Tri (TMEthyleneGlycol) benzoate or PFPTTEG) or by the ligand 2,2- (methoxyethoxy) acetylchloride (MEAC). For example, it may be the G3 DAB functionalized by the MEAC ligand (FIG. 10) or by the PFPTTEG dendron (FIG. 11).

[68] According to a particular embodiment of the invention, the water-soluble dendrimer may be a nona-ammonuim chloride dendrimer having the following structure:

which dendrimer is functionalized at its periphery by a solubilizing group corresponding to one of structures (II) or (III), preferably to structure (III), as follows:

Structure (II)

Structure (III) wherein: n, n ₁ , n ₂ and r ₃ independently represent an integer from 1 to 500, and

R 1, R 1, R ₂ and R ₃ independently represent C 1 -C 4 alkyl, C ₂ -C ₄ alkene, C ₂ -C ₄ alkyne, or optionally substituted C ₁ -C ₁₀ aryl.

[69] It may be noted that the nona-ammonuim chloride dendrimer is not soluble in water, hence the advantage of previously functionalizing it with solubilizing groups, for example with the PFPTTEG dendron, for the encapsulation of vitamin C. An example of functionalization methodology is given in the document by Pan et al., Macromolecules

(2000), 33, pages 3731-3738 (6). Indeed, this document describes that it is preferable to functionalize the anionic dendrimers with solubilizing and biocompatible groups even if they have a lower toxicity than cationic dendrimers as shown in the document.

Malik et al., J. Control. Release (2000), 65, p. 133 (7).

[70] Thus, according to a particular embodiment of the invention, the water-soluble dendrimer can respond to the structure described in FIG. 12 ("nona-amine" dendrimer functionalized by the PFPTTEG dendron). [71] According to one particular embodiment of the invention, the water-soluble dendrimer can make supramolecular bonds with the at least one molecule of vitamin C. [72] According to one particular embodiment of the invention, the at least one vitamin C molecule can be maintained at the heart or at the periphery of the at least one water-soluble dendrimer.

[73] Indeed, their multiple cavities and their reactive groups at the periphery, allow the recognition and capture of anions, such as the ascorbate anion. Thus, vitamin C can be contained in the heart or maintained on the surface of the dendrimer by supramolecular bonding, and especially by electrostatic bonding. Indeed, the ascorbic acid molecules have a pKa equal to 4.11 at 25 ° C in water, and thus form ammonium ascorbates with the amines of the dendrimers when these two products are in solution. In addition, the vitamin C molecules also carry out hydrogen bonds between its alcohol functions and the amines of the dendrimers. In the case of functionalized dendrimers, the PEG groups can participate in the transport of the ascorbic acid molecules either by carrying out hydrogen bonds with the ascorbate molecules, or by keeping them at the hydrophobic core of the dendrimer.

[74] A dendrimer can contain up to 500 molecules of vitamin C.

Thus, according to a particular embodiment of the invention, the average encapsulation number can be from 14 (± 1) to 500 (± 20) vitamin C molecules per dendrimer. In addition, this average encapsulation number can be adjusted according to the desired application. Indeed, depending on the amount of vitamin C added, we can encapsulate more or less up to a limit (which corresponds to the maximum number of vitamin C molecules that can be encapsulated). [75] The release of vitamin C can be carried out in water, for example by exchange with water molecules, where the ascorbate molecules can be protonated before being released in the form of ascorbic acid. The release can also take place in other solvents where vitamin C is soluble, such as ethanol. The release could be controlled by performing a dialysis to study the kinetics of release. In fact, the use of a porous semipermeable membrane (with pores of identical and known diameters) makes it possible to separate the vitamin C molecules from the dendrimers: by the effect of osmosis and molecular agitation, the small molecules, namely the Vitamin C molecules cross the membrane, while large molecules, the dendrimers, are retained inside the dialysis membrane. A quantification of these vitamin C molecules makes it possible to follow the kinetics of release. In addition, the release kinetics of vitamin C can be modified according to the pH since the pH influences the ratio between the number of ascorbic acid molecules and ascorbate.

[76] The invention also relates to a cosmetic composition comprising a conjugated dendrimer according to the invention.

[77] Indeed, the conjugated dendrimer according to the invention can be formulated in a cosmetic product to ensure a modulated delivery of vitamin C on the skin for protective (anti-radical) and / or regenerative purposes. [78] Thus, the invention also relates to a conjugated dendrimer according to the invention as an antioxidant and / or anti-inflammatory agent. [79] In addition, the invention also relates to the use of the conjugated dendrimer according to the invention for the preparation of a cosmetic composition, for example anti-aging or anti-wrinkle. [80] The invention also relates to a cosmetic care method comprising a step of applying to the skin a cosmetic composition comprising a conjugated dendrimer according to the invention.

[81] The invention also relates to a process for preparing a conjugated dendrimer comprising at least one water-soluble dendrimer encapsulating at least one vitamin C molecule, said method comprising a reaction step of said water-soluble dendrimer with said less one molecule of vitamin C in a solvent and at a temperature that facilitates the formation of supramolecular bonds of the dendrimer and the vitamin C molecule. It may be noted that water is the solvent which allows both solubility and formation links between the dendrimer and vitamin C.

[82] Indeed, the bringing together of these two species in a solvent leads instantly to the formation of supramolecular interactions. [83] According to a particular embodiment of the invention, during the preparation process, the combination of the dendrimer and the vitamin C molecule can proceed via at least one of the following bonds: Ammonium ascorbate with an amino function of the dendrimer, hydrogen bond formation between a hydroxyl function of vitamin C and an amino function of the dendrimer.

[84] According to the invention, the reaction step can be carried out in a solvent selected from the group comprising water, alcohols ¹ R ^S -OH where R ^s is a C1-C6 alkyl radical, for example 1 ethanol, or a mixture thereof. Indeed, the solvent used can solubilize partially or completely ascorbic acid and the dendrimer. For example, ascorbic acid is only partially soluble in ethanol. ^!

[85] According to the invention, the reaction step can be carried out during a reaction time of less than 1 minute, for example less than 30 seconds, for example less than 1 second. Indeed, the reaction time can range from a few milliseconds to a few seconds, preferably from a few milliseconds to a second.

[86] According to the invention, the reaction step can be carried out at a temperature ranging from 3 to 90 ^° C., preferably from 15 to 35 ^° C., preferably from 20 to 30 ^° C. In addition, an increase of temperature can help speed up the reaction rate. [87] According to the invention, the reaction step can be carried out at a pH i ranging from 0 to 14, preferably at a pH of 6 to 8, preferably at a pH of 7. In addition, the pH can influence the rate of reaction, but especially the encapsulation efficiency, indeed, the pH has an influence on the formation of hydrogen bonds between ascorbic acid and the dendrimer.

[88] According to a particular embodiment of the invention, the method of preparation may comprise reacting in water a sufficient amount of vitamin C to achieve a concentration of less than 10 ^Λ 2 mg / ml, with an amount sufficient dendrimer to obtain a conjugated dendrimer having a vitamin C: dendrimer ratio of 14 to 500, the reaction being carried out at a pH of 6 to 8, preferably 7, and at a temperature of 25 ° C +/- 10 ⁰ C preferably at a temperature of 25 ° C +/- 5 ° C. [89] Thus, the method of preparation has the advantage of using green chemistry, the encapsulation can be entirely carried out in water. This has advantages both in terms of environmental preservation, lower cost of production, but also purity, allowing applications in many fields, including the pharmaceutical and cosmetic fields.

[90] According to a particular embodiment of the invention, the method of preparation may further comprise, prior to the reaction step, a functionalization step, of functionalizing the periphery of the dendrimer with an organic surface agent selected from the group comprising: a pharmaceutical molecule, a targeting molecule or a solubilizing group.

[91] Other advantages may still appear to those skilled in the art on reading the examples below, illustrated by the accompanying figures, given for illustrative purposes.

Brief Description of the Figures - Figure 1 shows the DAB G2 dendrimer.

Figure 2 shows the DAB G3 dendrimer. Figure 3 shows the DAB G4 dendrimer. Figure 4 shows the DAB G5 dendrimer.

Figure 5 shows the PAMAM GO dendrimer.

Figure 6 shows the PAMAM G1 dendrimer.

Figure 7 shows the PAMAM G2 dendrimer. - Figure 8 shows the PAMAM G3 dendrimer.

Figure 9 shows the PAMAM G4 dendrimer.

Figure 10 shows the DAB G3 dendrimer functionalized by the MEAC.

FIG. 11 represents the DAB G3 dendrimer functionalized by the PFPTTEG.

Figure 12 shows the nona-amine dendrimer functionalized by PFPTTEG.

FIG. 13 represents the graphs of variation of the chemical shift δ (in ppm or parts per million) of the proton signals in ¹ H NMR relative to the number N of ascorbic acid molecules (AA) per dendrimer: (a) represents the graph of removal of proton signals 1 to 4 of the DAB G2 dendrimer (in ppm) relative to N; and (b) the proton signal shielding graph A to C of I ¹ AA (in ppm) relative to N. FIG. 14 shows the graphs of variation of the chemical shift δ (in ppm or part per million) of the proton signals in ¹ H NMR relative to the number N of ascorbic acid molecules (AA) by dendrimer: graph of removal (a) of the proton signals 1 to 4 of the DAB G3 dendrimer in ppm relative to N; shielding graph (b) of proton signals A to C of I ¹ AA in ppm relative to N.

FIG. 15 shows the graphs of variation of the chemical shift δ (in ppm) of the proton signals in ¹ H NMR relative to the number N of ascorbic acid molecules (AA) per dendrimer: (a) graph of removal of the signals of the protons 1 to 3 of the DAB G5 dendrimer in ppm relative to N; (b) shielding graph of proton signals A to C of AA in ppm relative to N. FIG. 16 represents the graphs of variation of the chemical shift δ (in ppm) of the proton signals in ¹ H NMR relative to the number N of ascorbic acid molecules (AA) per dendrimer: (a) graph of removal of the signals of the protons 1 to 4 and the 4 'of PAMAM G1 dendrimer in ppm relative to N; (b) shielding graph of proton signals A to C of the AA in ppm relative to the number N.

FIG. 17 represents the graphs of variation of the chemical shift δ (in ppm) of the proton signals in ¹ H NMR relative to the number N of ascorbic acid molecules (AA) per dendrimer: (a) graph of removal of the signals of the protons 1 to 4 and the 2 'of PAMAM G4 dendrimer in ppm relative to N; (b) shielding graph of proton signals A to C of AA in ppm relative to N.

FIG. 18 represents the graphs of variation of the chemical shift δ (in ppm) of the proton signals in ¹ H NMR relative to the number N of ascorbic acid molecules (AA) per dendrimer: (a) graph of removal of the signals of the protons 1 to 3 of the DAB G3 dendrimer functionalized by the MEAC ligand in ppm relative to N; (b) shielding graph of proton signals A to C of AA in ppm relative to N.

FIG. 19 represents the graphs of variation of the chemical shift δ (in ppm) of the proton signals in ¹ H NMR relative to the number N of ascorbic acid molecules (AA) by dendrimer: (a) proton signal deblinding 1 to 3 of the DAB G3 dendrimer functionalized by the PFPTTEG dendron in ppm relative to N; and (b) shielding the proton signals A to C of the AA in ppm relative to N. - Figure 20 represents the graph of variation of the chemical shift δ (or shielding) of the ¹ H NMR signals of the protons A to C AA in ppm relative to the N number of AA molecules per non-amine dendrimer functionalized by the PFPTTEG dendron. EXAMPLES

EXAMPLE 1 Functionalisation of the DAB G3 Dendrimer by the MEAC Monomer

The functionalization of the DAB G3 dendrimer by the MEAC monomer is carried out according to the following reaction, as described in the document by Kojima, C. et al. Bioconjugate Chem. (2000) 11, 910-917 (8):

Dendron 2,2- (methoxyethoxy) acetylchloride (210 mg: 1.38 mmol: 2 equiv. Per NH ₂ ) as well as triethylamine (185 mg: 1.84 mmol: 1.5 equiv. Per NH ₂ ) are added to a solution of DAB G3 (97 mg: 57.5 μmol) in DMF (1mL). The mixture is stirred for 24 hours at room temperature under nitrogen.

1 ml of distilled water is then added to the mixture which is allowed to stir for 10 minutes before concentrating the product under reduced pressure. It is diluted in 5 mL of dichloromethane before being extracted into an aqueous solution of 1% potassium carbonate. The product is then purified by recrystallization in pentane and then by a column of chromatography (SiO ₂ ) with chloroform / methanol (95/5) as eluent.

50 mg (25%) of functionalized dendrimer is obtained.

¹ H NMR (CDCL ₃ , 250 MHz); δ ppm: 1.67 (m, CH ₂ -CH ₂ -N); 2.43 (m, CH ₂ -N); 3.31 (s, CH ₂ -NH); 3.39 (s, CH ₃ -O); 3.58 (m, CH ₂ -CH ₂ -O); 3.97 (m, CH ₂ -CO); 7.34 (s, NH-CO).

Example 2: Synthesis of the PFPTTEG dendron

The synthesis of pentafluorophenyl tris 3,4,5-tri (triethyleneoxy) benzoate dendron, described in Baars, M.W.P.L. et al., Angew. Chem. Int. Ed. (2000), 39 (7), 1285-1288 (9), is carried out according to the following succession of reaction steps: - Synthesis of monomethyltriethylene glycol monotosylate Synthesis of tris 3,4,5-tri (triethyleneoxy) acid ) benzoic Synthesis of pentafluorophenyl tris 3,4,5-tri (triethyleneoxy) benzoate

Synthesis of monomethyltriethylene glycol monotosylate: The monomethyltriethylene glycol monotosylate is synthesized according to the following reaction:

CH ₂ Cl ₂ / And ₃ N

To a solution of monomethyl triethylene glycol (16.416 g: 99.775 mmol) in CH ₂ Cl ₂ (10 mL) was added triethylamine (199.95 mmol: 2 equiv) and p-toluenesulfonyl chloride (TsCl: 149). 96 mmol: 1.5 equiv). After stirring for 24 hours, the mixture is washed twice with NaHCO ₃ aq. The organic phase is dried using anhydrous sodium sulfate, filtered and evaporated in vacuo. The residue is purified in a column chromatography (SiOa), and eluted with a mixture of petroleum ether / ethyl acetate.

We obtain 17.8 g (56%) of monomethyltriethylene glycol monotosylate. ¹ H NMR (CDCL ₃ , 250 MHz); δ ppm: 2.45 (s, CH ₂ -C _arom ); 3.37 (s,

CH ₃ -O); 3.59 (t, CH ₂ -O); 4.16 (t, CH ₂ -OS); 7.36 (d, CH _aro mC-CH ₃ ); 7.78 (d,

CH arom-C-o).

Synthesis of tris-3,4,5-tri (triethyleneoxy) benzoic acid: Tris 3,4,5-tri (triethyleneoxy) benzoic acid is synthesized according to the following reaction: a) K ₂ CO ₃ / acetone b) LiOKH ₂ O / MeOH

To a solution of methyl ester trihydroxybenzoate (0.329 g: 1.79 mmol) in acetone (10 mL) is added potassium carbonate (1.772 g: 0.018 mol: 3.5 equiv.) And monotosylate monomethyltriethylene glycol (2 g: 0.006 mol: 3.5 equiv). The heterogeneous mixture is refluxed for 24 hours under nitrogen; the pink mixture obtained is then filtered to get rid of the excess potassium carbonate. The acetone is then evaporated and the product is dissolved in dichloromethane (12 mL). Three washings followed with 10 mL of water, then 5 mL of 1M HCl and finally 10 mL of water, before evaporating the solvent from the organic phase. The product is then purified in a chromatography column (SiO ₂ ) with, as eluent, the chloroform / methanol 97: 3 mixture.

8 g (80%) of methyl ester tris 3,4,5-tri (triethyleneoxy) benzoate are then obtained.

¹ H NMR (CDCL ₃ , 250 MHz); Δ ppm: 3.37 (s, CH ₃ -O); 3.65 (m, CH ₂ -O); 3.88 (s, CH ₃ -O); 4.19 (s, CH ₂ -OC _ar om); 7.29 (s, CH _a rom). The methyl ester tris 3,4,5-tri (triethyleneoxy) benzoate (3 g: 4,823 mmol) is added to a solution of LiOH (251 mg: 6.029 mmol: 1.25 equiv) in a water / methanol mixture (10 g). mL, 1/3 v / v), before mixing for 12 hours. The mixture is evaporated under vacuum, redissolved in water and extracted with dichloromethane to remove the salts.

2.4 g (80%) of tris 3,4,5-tri (triethyleneoxy) benzoic acid are obtained.

¹ H NMR (CDCL ₃ , 250 MHz); δ ppm: 3.32 (s, CH ₃ -O); 3.61 (m, CH ₂ -O); 4.00 (s, CH ₂ -O-Carom); 7.26 (s, CHarom).

Synthesis of pentafluorophenyl tris 3,4,5-tri (triethyleneoxy) benzoate:

Pentafluorophenyl tris 3,4,5-tri (triethyleneoxy) benzoate is synthesized according to the following reaction: DCC / Diglyme

A solution of 3,4,5-tri (triethyleneoxy) benzoic acid tris (2.4 g: 3.846 mmol) and pentafluorophenol (757 mg: 4.115 mmol: 1.07 equiv) was prepared in diglyme (4 mL). To this homogeneous solution cooled to 0 ^° C., 1,3-dicyclocarbodiimide (DCC) (889 mg: 4.308 mmol: 1.12 equiv) is added. After the complete addition of DCC, the reaction medium is allowed to return to room temperature. After 24 hours, the reaction mixture is filtered and this filtrate is concentrated under reduced pressure. The product is finally precipitated with cyclohexane and purified by a chromatography column (SiO ₂ ) with chloroform / methanol (9: 1) as eluent.

We obtain 1.7 g (56%) of pentafluorophenyl tris 3,4,5-tri (triethyleneoxy) benzoate.

¹ H NMR (CDCL ₃ , 250 MHz); δ ppm: 3.32 (s, CH ₃ -O-CH ₂ ); 3.61 (m, CH ₂ -O); 4.23 (m, CH ₃ -OC); 7.44 (s, CH _arom ). Example 3: Functionalization of the DAB G3 Dendrimer by the PFPTTEG Dendron

The functionalization of the DAB G3 dendrimer by the PTPTTEG dendron is carried out according to the following reaction:

To a solution of DAB G3 (12 mg: 7.114 μmol) in dichloromethane (8 ml) was added dendron pentafluorophenyl tris 3,4,5-tri (triethyleneoxy) benzoate (92 mg: 116.67 μmol: 1.025 equiv. by NH ₂ ). The mixture is stirred for 12 hours before being extracted with 0.1M NaOH. After evaporation of the aqueous phase, the product is dissolved in dichloromethane and precipitated by addition of petroleum ether.

A few milligrams of product are obtained in sufficient quantity to carry out the titration.

¹ H NMR (CDCl ₃ , 250 MHz); δ ppm: 1.72 (m, CH ₂ -CH ₂ -NH); 2.46 (m, CH ₂ -N); 2.96 (m, CH ₂ -NH); 3.45 (s, CH ₃ -O); 3.65 (m, CH ₂ -CH ₂ -O); 4.15 (m, CH ₂ -O-Carom) 5.43 (s, NH-CO); 7.05 (s, CH _arom ). Example 4: Functionalization of the Nona-Amine Dendrimer by the PFPTTEG Dendron

The functionalization of the non-amine dendrimer is carried out according to the following reaction:

The non-amine dendrimer (50 mg: 31 μmol) and the dendron

PFPTTEG (226 mg: 286 μmol: 1.025 equivalents per amine) are dissolved in 50 ml of distilled dichloromethane, before adding triethylamine (56 mg: 558 μmol: 2 equivalents per amine). The mixture is stirred under nitrogen at room temperature for 12 hours.

The mixture is dried under vacuum, then the product is dissolved again in dichloromethane before being extracted with 150 ml of a 0.1M solution of NaOH. It is then washed 3 times with pentane and then purified by recrystallization in the reaction mixture. dichloromethane with pentane.

112 mg (55%) of non-amine dendrimer functionalized by the dendron PFPTTEG is obtained.

¹ H NMR (CDCL ₃ , 250 MHz); δ ppm: -0.13 (s, CH ₃ -Si); 0.54 (s,

CH ₂ -Si); 1, 08 (s, CH ₂ -CH ₂ -Si); 1.87 (s, CH ₂ -CH ₂ -CH ₂ -Si); 2.85 (s, Si-CH ₂ -N); 3.32 (s, CH ₃ -O); 3.62 (s, CH ₂ -CH ₂ -O); 4.13 (s, CH ₂ -OC _from ndron); 6.96 (s,

CHarom dedrimer)! 7, 18 (S, CHarom dendron) -

IR: v = 3320 cmτf ¹ and v = 1625 cm- ¹ (NH-CO) Elemental analysis: Calculated: C, 57.33; H, 8.57; N, 91; O, 28.36; If 3.83; Found: C, 55.07; H, 8.55.

Example 5 Encapsulation of Vitamin C Using a Few Commercial Dendrimers

Five AA titrations were performed: three with DAB generational 2 (DAB G2), 3 (noted DAB G3) and 5 (DAB G5), and two others with PAMAM generation 1 dendrimers (noted PAMAM). G1) and 4 (noted PAMAM G4).

The interpretation of the NMR data will be detailed in the case of a single generation for each dendrimer (DAB G2 and PAMAM G4); for the other titrations, a diagram of the dendrimer as well as two summary graphs of the NMR data will be presented. A final table summarizing all the results obtained will close this example.

It should be noted that the following reactions are analyzed immediately performed (about ten seconds separates the two operations) which is an additional advantage for the future cosmetic use of the conjugate because its instantaneous synthesis would optimize the formulation time.

a) The G2 ATM

Operating protocol for encapsulation of vitamin C in DAB G2: Fifteen E samples (numbered from 1 to 15) with increasing concentrations of ascorbic acid (AA) relative to the dendrimer

DAB G2 (Figure 1) were prepared as follows:

An S1 solution of ascorbic acid is obtained by dissolving 170.7 mg of ascorbic acid (AA) in 1.5 ml of D ₂ O. The ascorbic acid used here is sold under the reference 255564 by Sigma-Aldrich.

(France). Each sample E was prepared by adding a volume V of S1 solution of ascorbic acid in an NMR tube containing 5 mg of DAB G2 dendrimer in 300 μl of D2O. This dendrimer is marketed under the reference 679895 by Sigma-Aldrich (France).

The volume V introduced, the ratio of the number of AA molecules introduced by DAB G2 dendrimer, and the mass of AA introduced into the NMR tube for each sample are detailed in Table 2 below.

Table 2

Titration of ascorbic acid contained in DAB G2 and results:

The previous samples (E1 to E15) were analyzed directly after adding the ascorbic acid solution without additional treatment or waiting time because the encapsulation of vitamin C in the dendrimer is instantaneous. The NMR experiments were carried out by the BRUKER AC250FT spectrometer at 25 ° C.

The data of the fifteen samples analyzed by NMR are detailed in the graphs presented in FIG. Chart (a) is a representation of the various ppm NMR data for all the hydrogens of the DAB G2 dendrimer as a function of the amount of ascorbic acid added. The protons of the dendrimer are numbered from 1 to 4 according to the diagram below, to better visualize the displacement of the NMR signals of each proton:

Thus, curve 1 gives the chemical shift δ in ¹ H NMR of the hydrogen numbered 1 for each of the samples E1 to E15.

The NMR data of the peak corresponding to the protons of the primary amines are not accessible; in fact, the NMR spectra are carried out in the solvent D ₂ O, and these protons are in continuous exchange with the solvent, hence the absence of their signals.

DAB G2 signals are progressively deblocked to a concentration of about 30 AA / DAB, which means that all primary and tertiary amines quaternized with AA, and some additional AAs have bound themselves to the dendrimer probably by hydrogen bonds with the protons of the primary amines at the periphery of the dendrimer. In addition, the AAs first settle on the periphery because curves 1 and 2 have an initial slope greater than those of curves 3 and 4.

Chart (b) is the various NMR data in ppm of ascorbic acid hydrogens (AA) indexed from A to C as in the diagram below:

From 14 AA / DAB peaks of AA begin to blister which corresponds to the appearance of ascorbic acid in the solution. So we are in the presence of a mix ascorbic acid / ascorbate. However, curve A, which is the most significant, continues to grow to a concentration of about 60 AA / DAB. So there would be an AA / ascorbate balance when the added AA number is between 14 and 60. Then, the curve stagnates, which means that beyond 60 AA added one would be in the presence of a dynamic equilibrium . In addition it is also possible to calculate the number of ascorbic acid molecules attached to the dendrimer by means of the NMR data because, when the peak of the proton A of ascorbic acid is halfway between the values of this same proton under the ascorbic and ascorbate form (ie δ = 4.7 ppm), then we can consider that half of the ascorbic acid molecules that can be transported is fixed. This dendrimer would allow us to carry up to 60 AA.

It can be noted that a titration of propionic acid was also carried out with the DAB G2 dendrimer to approve the existence of encapsulation and hydrogen bonds between the AA and the dendrimer. Indeed, the results obtained show the fixation of only 8 propionic acids, which corresponds to the formation of 8 ammonium propionates at the periphery of the dendrimer.

The alcohol functions present on the ascorbic acid would then form hydrogen bonds with the amines of the dendrimer and would also allow them to encapsulate in his heart.

b) The G3 ATM Operating protocol of encaosulation of vitamin C in DAB G3:

Twelve samples (E1 to E12) with increasing ascorbic acid (AA) concentrations relative to the DAB G3 dendrimer (Figure 2) were prepared as follows:

An S2 solution of ascorbic acid is obtained by dissolving 104.5 mg of ascorbic acid (AA) in 1 ml of D ₂ O.

Each sample E was prepared by adding a volume V of solution S2 of ascorbic acid in an NMR tube containing 10 mg of DAB G3 dendrimer in 300 μl of D ₂ O. This dendrimer is marketed under the reference 469076 by the company Sigma. AIdrich (France).

The amounts of AA introduced and the ratio of the number of AA molecules introduced by DAB G3 dendrimer into the NMR tube for each sample are detailed in Table 3 below.

The twelve samples were then analyzed directly by NMR. NMR data are detailed in the graphs shown in Figure 14.

Graph (a) shows the removal of the dendrimer hydrogens numbered from 1 to 4 as below:

Chart (b) shows the various NMR data in ppm of the ascorbic acid hydrogens (AA) indexed from A to C as below:

A reasoning similar to that of the previous point a) makes it possible to conclude that, for the DAB G3 dendrimer, 16 molecules of ascorbate can be fixed before the appearance of the ascorbic acid and that the DAB G3 dendrimer can comprise 80 molecules of Ascorbate fixed.

c) The DAB Gδ

Operating protocol for encapsulation of vitamin C in DAB G5:

Fifteen samples (E1 to E15) comprising increasing amounts of ascorbic acid (AA) relative to the DAB G5 dendrimer (Figure 4) were prepared as follows:

An S3 solution of ascorbic acid is obtained by dissolving 91.5 mg of ascorbic acid (AA) in 1.5 ml of D2O.

Each sample E was prepared by adding a volume V of solution S3 of ascorbic acid in an NMR tube containing 5 mg of DAB G5 dendrimer in 300 μl of D ₂ O. This dendrimer is marketed under the reference 469092 by Sigma AIdrich (France).

The amounts of AA introduced and the ratio of the number of AA molecules introduced by DAB G5 dendrimer into the NMR tube for each sample are detailed in Table 4 below.

The fifteen samples were analyzed by NMR. NMR data are detailed in the graphs shown in Figure 15.

Chart (b) shows the various NMR data in ppm of ascorbic acid hydrogens (AA) indexed from A to C above.

d) PAMAM G1 dendrimer

Protocol for the encapsulation of vitamin C in PAMAM G1:

Fifteen samples (E1 to E15) comprising increasing amounts of ascorbic acid relative to the PAMAM G1 dendrimer (FIG. 6) were prepared as follows:

An S4 solution of ascorbic acid is obtained by dissolving 61.5 mg of ascorbic acid (AA) in 1 mL of D ₂ O.

Each sample E was prepared by adding a volume V of S4 solution of ascorbic acid in an NMR tube containing 5 mg of dendrimer PAMAM G1 in 300 μl of D ₂ O. This dendrimer is marketed under the reference 597414 by Sigma-Aldrich (France).

The amounts of AA introduced and the ratio of the number of AA molecules introduced by PAMAM G1 dendrimer into the NMR tube for each sample are detailed in Table 5 below.

The fifteen samples were analyzed by NMR. NMR data are detailed in the graphs shown in Figure 16.

e) PAMAM G4 Dendrimer

Protocol for the encapsulation of vitamin C in PAMAM G4:

Fifteen samples (E1-E15) comprising increasing amounts of ascorbic acid relative to the PAMAM G4 dendrimer (FIG. 9) were prepared as follows:

An S5 solution of ascorbic acid is obtained by dissolving 62 mg of ascorbic acid (AA) in 2 ml of D ₂ O.

Each sample E was prepared by adding a volume V of solution S5 of ascorbic acid in an NMR tube containing 5 mg of PAMAM G4 dendrimer in 300 μl of D ₂ O. This dendrimer is marketed under the reference 597856 by Sigma. AIdrich (France).

The amounts of AA introduced and the ratio of the number of AA molecules introduced by PAMAM G4 dendrimer into the NMR tube for each sample are detailed in Table 6 below.

Table 6.

Titration of ascorbic acid contained in PAMAM G4 and results:

The fifteen samples were analyzed by NMR. NMR data are detailed in the graphs shown in Figure 17.

PAMAM G4 signals are progressively deblocked to a concentration of 64 AA / PAMAM. It can be thought that the AA is initially encapsulated in the dendrimer, which generates only a weak removal. Then the signals 2 and 3 corresponding to the primary primary amines and internal tertiary amines are strongly deblinded, hence the presence of ammonium ascorbate in the heart and the periphery of the dendrimer.

This graph does not allow us to consider preference for AA binding on primary or tertiary amines because the curves evolve similarly. It is also possible that the AA is fixed progressively from generation to generation, the steric hindrance thus slowing down its progress towards the heart of the dendrimer.

The fixation of AA by the dendrimer stagnates and the curves then form a plateau when the concentration reaches 400 AA / PAMAM; this suggests that most of the amines of the PAMAM dendrimer have quarreled and that some additional AAs have attached to the dendrimer either by encapsulation in its pores or by hydrogen bonding with the protons of the primary amines at its periphery.

Up to a concentration of 64 AA / PAMAM, the peaks of AA are unblocked, then they begin to bind, which might suggest a first fixation of AA on 64 primary external amines.

Then several hundred molecules of ascorbic acid would seem to attach to the heart of the dendrimer and the periphery by hydrogen bonds, while others remain in the acid form in solution.

Finally, the fact that peaks 1 and 2 dissociate respectively in

V and 2 'can be explained by the number of generations of the dendrimer. In fact, the amines react differently with the AA according to the generation to which they belong, this being explained by a congested access according to the depth of the amine at the heart of the dendrimer.

f) Conclusion

The following table 7 summarizes the results of all the previous titrations by giving the number of ammonium ascorbates formed before and after appearance of ascorbic acid in the medium:

Table 7.

The results obtained are consistent with those expected; indeed, the greater the generation of the dendrimer, the greater the number of ascorbic acid molecules transported is important. In addition, it should be noted that the nature of the dendrimer (DAB or PAMAM) has no influence on the fixation because the results are identical in both cases; only the number of primary and tertiary amines present at the periphery and at the heart of the dendrimer are important (let us recall that the DAB G2 and PAMAM G1 dendrimers, as well as the

DAB G5 and PAMAM G4 have an equivalent number of amino functions).

The desired objective, which was to visualize the existence of bonds between vitamin C and dendrimers, is therefore achieved. It now remains to carry out the same type of experiments as well as other complementary ones in order to optimize the result obtained and to find the right compromise between transport efficiency, solubility and toxicity.

Example 6 Encapsulation of Vitamin C Using Some Functionalized Dendrimers The various advantages provided by a polyethylene glycol chain graft at the periphery of a molecule have already been demonstrated many times. Indeed, this water-soluble, non-toxic and non-immunogenic polymer is very suitable for biological use thanks to its high water solubility and biocompatibility.

It has therefore been wise to functionalize the dendrimers with two different molecules containing either one (MEAC monomer) or three PEG chains (PFPTTEG dendron) in order to compare the properties provided by each of them.

a) DAB G3 dendrimer functionalized with MEAC monomer

The functionalization of the DAB G3 dendrimer with the MEAC monomer is carried out according to the synthesis described in Example 1.

Operating protocol for encapsulation of vitamin C:

Fourteen samples (E1 to E14) comprising increasing amounts of ascorbic acid relative to the DAB G3 dendrimer functionalized with the MEAC monomer (FIG. 10) were prepared as follows:

An S6 solution of ascorbic acid is obtained by dissolving 49 mg of ascorbic acid (AA) in 2 mL of D ₂ O.

Each sample E was prepared by adding a volume V of solution S6 of ascorbic acid in an NMR tube containing 5 mg of DAB G3 dendrimer functionalized with the monomer MEAC in 300 μl of D ₂ O.

The amounts of AA introduced and the ratio of the number of dendrimer-introduced AA molecules in the NMR tube for each sample are detailed in Table 8 below.

Table 8.

Titration of ascorbic acid contained in the dendrimer and results:

NMR titration of ascorbic acid with this functionalized dendrimer is performed to compare its vitamin C transport properties. The NMR data are detailed in the graphs shown in Figure 18.

From the results obtained, it is clear that this dendrimer carries the transport of vitamin C, with a yield similar to the non-functional DAB G3 dendrimer but with the advantages of much better solubility and biocompatibility. Indeed, this dendrimer can fix 16 molecules of ascorbate before appearance of ascorbic acid molecules, and up to 80 molecules of ascorbate before reaching a dynamic equilibrium. As for the NMR data of the dendron (5, 6 and 7), they do not vary at all during the titration; this means that monoethylene glycol branches do not participate in AA binding or encapsulation within the dendrimer. b) The DAB G3 dendrimer functionalized by the PFPTTEG dendron

The PFPTTEG dendron is first synthesized according to Example 2 in order to perform the functionalization of the DAB G3 dendrimer according to the protocol described in Example 3.

Operative protocol of encaosulation of vitamin C:

Fourteen samples (E1 to E14) comprising increasing amounts of ascorbic acid relative to the DAB G3 dendrimer functionalized with the PFPTTEG dendron (FIG. 11) were prepared as follows:

An S7 solution of ascorbic acid is obtained by dissolving 15.8 mg of ascorbic acid (AA) in 2 ml of D2O. Each sample E was prepared by adding a volume V of S7 solution of ascorbic acid in an NMR tube containing 5 mg of DAB G3 dendrimer functionalized with the PFPTTEG dendron in 300 μl of D ₂ O.

The amounts of AA introduced and the ratio of the number of dendrimer-introduced AA molecules in the NMR tube for each sample are detailed in Table 9 below.

Table 9.

Titration of ascorbic acid contained in the dendrimer

NMR titration of AA is performed to evaluate the transport capacity of vitamin C by the DAB G3 dendrimer functionalized with the PFPTTEG dendron.

The dendrimer obtained is soluble in a wide range of more apolar solvents such as toluene and dichloromethane more polar such as water, methanol and acetonitrile. In addition, this functionalized dendrimer is soluble in water in all proportions, which is an additional advantage allowing a high yield of AA transported.

NMR titration of ascorbic acid with this functionalized dendrimer is performed to compare its vitamin C transport properties. NMR data are detailed in the graphs shown in Figure 19.

From the results obtained, it is obvious that the functionalization of the dendrimer would in no way hinder the transport of vitamin C, on the contrary. Indeed, this dendrimer can fix 45 molecules of ascorbate before appearance of ascorbic acid molecules, and up to 100 molecules of ascorbate thereafter, these numbers being significantly higher than those obtained by the same non-functional DAB G3 dendrimer . The dendron thus makes it possible to make connections with the AA, and participates greatly in a better encapsulation of AA in the heart of the dendrimer, property ignored at the beginning.

It therefore appears that this new dendrimer has all the advantages of good biocompatibility, good solubilization in water and excellent AA transport efficiency, the last property being an added advantage over dendrimer functionalized with MEAC monomer.

c) The "nona-amine" dendrimer functionalized by the PFPTTEG dendron The PFPTTEG dendron is first synthesized according to Example 2 in order to carry out the functionalization, according to the protocol described in Example 4, of a new nine-branched water-soluble dendrimer, named here the non-amine dendrimer. .

Operative protocol of encaosulation of vitamin C:

Fourteen samples (E1 to E14) comprising increasing amounts of ascorbic acid relative to the "nona-amine" dendrimer functionalized with the PFPTTEG dendron (FIG. 12) were prepared as follows:

An S8 solution of ascorbic acid is obtained by dissolving 26.7 mg of ascorbic acid (AA) in 2 ml of D2O.

Each sample E was prepared by adding a volume V of S8 solution of ascorbic acid in an NMR tube containing 5 mg of "nona-amine" dendrimer functionalized by the PFPTTEG dendron in 300 μL of D2O. The amounts of AA introduced and the ratio of the number of dendrimer-introduced AA molecules in the NMR tube for each sample are detailed in the following Table 10.

Table 10.

Titration of ascorbic acid contained in the dendrimer and results:

NMR titration of AA is performed with this new water-soluble functionalized dendrimer to identify the part of TEG chains of responsibility in the transport of vitamin C. The results are given in the proton deblinding graph of the AA presented in Figure 20.

Thanks to this dendrimer, which is functionalized with PFPTTEG dendrons, it is possible to transport more than one hundred AAs.

This molecule comprises 27 TEG chains, these latter create electrostatic bonds and a steric conformation favorable to the maintenance of the ascorbic acid molecules within the molecule, thus allowing their transport. This dendrimer, being composed of no primary or tertiary amine, shows us a chemical composition and an ideal conformation for the encapsulation of vitamin C molecules thanks to its particular properties provided by its heart and its dendrons. d) Conclusion

Ascorbic acid, whose exogenous contribution is essential for the human body, has the potential to be transported by certain organic molecules through a cosmetic product. Indeed, three types of vectors have been tested, namely pure commercial dendrimers, these same dendrimers functionalized with ethylene glycol chains, as well as new water-soluble dendrimers also comprising ethylene glycol chains whose synthesis is entirely achieved. in the laboratory.

The dendrimers and their particular properties allow excellent transport efficiency of the ascorbic acid molecules, especially when they are functionalized with a dendron comprising three triethylene glycol chains. The latter in fact perform hydrogen bonds with the ascorbic acid molecules and also allow them to encapsulate in the heart of the dendrimer, while providing solubility properties in water (as well as in a large number of solvents), biocompatibility, and especially stability. Thus, the PFPTTEG dendron clearly participates in a better encapsulation of the vitamin C molecules and also carries out hydrogen bonds with these same molecules which are then viewable by NMR. On the other hand, the chain of the MEAC group, three times shorter than that of the PFPTTEG group, does not allow to see, in NMR, a participation of the MEAC group on the encapsulation of vitamin C molecules.

The transport yields of the new water-soluble dendrimers functionalized with PEG chains differ according to whether the dendron is more or less imposing with longer or shorter chains. The two dendrimers functionalized with the PFPTTEG dendron lead to a better transport efficiency; indeed, thanks to their steric hindrance at the periphery of the dendrimers, they make it possible to keep the AA molecules inside the dendrimers, without interfering with the passage of AA molecules to his heart.

At the level of the transport efficiency of the vitamin C molecules, the best performing dendrimer is the one whose synthesis is entirely carried out in the laboratory, namely the nona-amine functionalized by nine dendrons, ie 27 ethylene glycol chains. In addition, this dendrimer does not contain internal amines, should have a lower toxicity than those marketed.

To conclude, dendrimers appear to be suitable for the transport of ascorbic acid molecules in cosmetics thanks to their controlled multivalence which can be used to attach one or more substances (drugs, enzymes, contrast agents for medical imaging). , ... here vitamin C) as well as targeting and solubilizing groups on the periphery. In addition, this type of vector has the advantage of being able to be synthesized in different sizes by varying its generation. Finally, since dendrimers are molecules with a well-defined structure (polymolecularity equal to one), they could provide a reproducible pharmacokinetic substance, a definite advantage for their future in application.

List of references

(1) Tomalia, DA; Baker, H.; Hall, M.; Kallos, G.; Martin, S.; Roeck, J.; Smith, P. Macromolecules (1986) 19, 2466.

(2) Astruc, D.; Research Avenues on Dendrimers towards Molecular Biology from Biomimation to Medicinal Engneering; CR. Acad. Sci.

(1996), 322, Series 2b, 757-766.

(3) Vandamme, Th. F.; Brobeck, L.; J.Control. Release (2005), 102 (1), 23-38.

(4) Zhuo R.X. ; B.; Lu Z.R. ; J. Control. Release (1999) 57, 249 (5) Liu, M.; Kono, K.; Frechet, J. M. J.; J. Polym. Sci. : part A: Polym. Chem. (1999) 3492.

(6) Pan, Y .; Ford, WT. ; Amphiphilic Dendrimers with both octyl and triethylenoxy methylether chain ends, Macromolecules (2000), 33, 3731-3738. (7) Malik, N.; Wiwittanapatapee, R.; Klopsh, R.; Lorenz, K.; Frey, H.; Weener, J.W. et al., J. Control. Release (2000), 65, 133.

(8) Kojima, C.; Kono, K.; Maruyama, K.; Takagishi, T.; Synthesis of Polyamidoamine Dendrimers with Poly (Ethylene Glycol) Grafts and their Ability to Encapsulate Anticancer Drugs. Bioconjugate Chem. (2000) 11, 910-7.

(9) Baars, M.W.P.L. et al., In The localization of water-soluble guests Poly (propylene imine) dendrimers, Angew. Chem. Int. Ed. (2000), 39 (7), 1285-1288.

Claims

A conjugated dendrimer comprising at least one water-soluble dendrimer encapsulating at least one molecule of vitamin C.

The conjugated dendrimer of claim 1, wherein said at least one dendrimer has a symmetrical radial structure.

The conjugated dendrimer of claim 1 or 2, wherein said at least one dendrimer has a globular structure.

The conjugated dendrimer of any one of claims 1 to 3, wherein said at least one dendrimer has a diameter of from 10 A to 50 A.

5. The conjugated dendrimer according to any one of claims 1 to 4, wherein said at least one dendrimer has at its periphery mainly free primary amino functions or ammonium functions.

The conjugated dendrimer of any one of claims 1 to 5, wherein said at least one dendrimer has one of the following structures:

10

60

The conjugated dendrimer of any one of claims 1 to 6, wherein said at least one dendrimer is functionalized at its periphery with at least one organic surface agent.

The conjugated dendrimer of claim 7, wherein said at least one organic surfactant is selected from the group consisting of: a pharmaceutical molecule, a targeting molecule or a solubilizing group.

The conjugated dendrimer of claim 8, wherein the organic surface agent is a polyethylene glycol chain solubilizing group.

The conjugated dendrimer of claim 9, wherein the solubilizing group has one of the following structures (II) or (III):

Structure (II)

Structure (III) wherein: n, n ₁ , n ₂ and n ₃ independently represent an integer from 1 to 500, and

R 1, R 1, R ₂ and R ₃ independently represent C ₁ to C ₆ alkyl, C 2 to C ₆ alkene, C 2 to C ₆ alkyne, or optionally substituted C ₆ to C 10 aryl.

11. The conjugated dendrimer according to claim 10, wherein the solubilizing group corresponds to one of the following structures (IV) or (V):

Structure (IV)

in which n is 1 and n 1, and n ₃ represent 3.

The conjugated dendrimer of any one of claims 1 to 5, wherein said at least one water-soluble dendrimer has the following structure:

Θ which dendrimer is functionalized at its periphery by a solubilizing group responding to one of the following structures (II) or (III):

Structure (II)

Structure (III) in which:

n, n, n ₂ and r 13 independently represent an integer of 1 to 500, and

R 1, R 1, R 2 and R ₃ independently represent a C ₁ to C ₆ alkyl, a C ₂ to C ₆ alkene, a C ₂ to C ₆ alkyne, or an optionally substituted C ₆ to C ₁₀ aryl.

The conjugated dendrimer of claim 12, wherein said at least one water-soluble dendrimer has the following structure:

The conjugated dendrimer of any one of claims 1 to 13, wherein said at least one water soluble dendrimer provides supramolecular linkages with the at least one vitamin C molecule.

The conjugated dendrimer of any one of claims 1 to 14, wherein said at least one vitamin C molecule is maintained at the heart or periphery of the at least one water-soluble dendrimer.

The conjugated dendrimer of any one of claims 1 to 15, wherein the average encapsulation number is 14 (± 1) to 500 (± 20) vitamin C molecules per dendrimer.

Cosmetic composition comprising a conjugated dendrimer according to any one of claims 1 to 16.

The conjugated dendrimer of any of claims 1 to 16 as an anti-oxidant and / or anti-inflammatory agent.

19. Use of the conjugated dendrimer according to any one of claims 1 to 16 for the preparation of an anti-aging or anti-wrinkle cosmetic composition.

20. A cosmetic care method comprising a step of applying to the skin a cosmetic composition comprising a conjugated dendrimer according to any one of claims 1 to 16.

A process for preparing a conjugated dendrimer comprising at least one water-soluble dendrimer encapsulating at least one vitamin C molecule, said method comprising a step of reacting said water-soluble dendrimer with said at least one molecule of vitamin In a solvent and at a temperature which facilitates the formation of supramolecular bonds of the dendrimer and the vitamin C molecule.

22. The method of claim 21, wherein the combination of the dendrimer and the vitamin C molecule proceeds via at least one of the following bonds: formation of ammonium ascorbate with an amine function of the dendrimer, Hydrogen bond formation between a hydroxyl function of vitamin C and an amine function of the dendrimer.

23. The method of claim 21 or 22, comprising reacting in water a sufficient amount of vitamin C to achieve a concentration of less than 10 ^Λ 2 mg / ml, with a sufficient amount of the dendrimer to obtain a conjugated dendrimer having a vitamin C: dendrimer ratio of 14 to 500, the reaction being carried out at a pH of 6 to 8 and at a temperature of 25 ° C. ± 5 ^° C.

The method of any one of claims 21 to 23, further comprising a functionalizing step prior to the vitamin C reaction step of functionalizing the periphery of the dendrimer with an organic surface agent selected from the group comprising: a pharmaceutical molecule, a targeting molecule or a solubilizing group.