WO2013160589A1

WO2013160589A1 - Biodegradable nanostructured materials, process for preparing them, and their use for the transport and release of substances of interest

Info

Publication number: WO2013160589A1
Application number: PCT/FR2013/050862
Authority: WO
Inventors: Ahmed MOURCHID; Corine Gerardin; Milad AL HELOU
Original assignee: Centre National De La Recherche Scientifique; Universite Montpellier 2 Sciences Et Techniques; Ecole Normale Superieure De Chimie De Montpellier
Priority date: 2012-04-27
Filing date: 2013-04-18
Publication date: 2013-10-31
Also published as: FR2989973B1; FR2989973A1

Abstract

The invention relates to biodegradable nanostructured materials, to a process for preparing them, and to their use for the transport and release of substances of interest. These materials are obtained in the form of thick films by mixing of a biopolymer and of micelles used as templates to generate pores. The elimination of said micelles makes it possible to free pores, which are intended to receive molecules of active principle by encapsulation, for the purpose of vectorization for human or animal use.

Description

Nanostructured biodegradable materials, process for their preparation and their use for the transport and release of substances of interest

The present invention relates to biodegradable nanostructured materials, process for their preparation and their use for the transport and release of substances of interest.

The development of biodegradable and implantable biomaterials for drug transport is of considerable interest as these materials allow for minimally invasive and finite duration applications.

Ideally, the release of the molecules constituting the active principle contained in the vectorized drugs, must be controlled so as to deliver a continuous dose locally. However, this is not the case in most materials designed for the transport of drugs, the diffusion of molecules being governed by a Fikin law, and therefore dependent on the concentration of molecules. Indeed, so far, the release is essentially induced by the erosion of the container and not by diffusion into pores. Therefore, in most of the vectors developed so far, the burst effect is often unavoidable and is opposed to a controlled and continuous diffusion of active molecules. It is therefore necessary to conduct studies on the design of appropriate biomaterials for diffusion control. Ideally, the nanostructuration should make it possible to conceive pores whose size is of the same order of magnitude as the size of the active molecules, thus allowing a controlled diffusion by single file of the encapsulated molecules.

It is also desirable to be able to develop these biomaterials without resorting to aggressive methods of treatment, physical or chemical, usually used in inorganic nanostructuring processes. Indeed, commonly used templates or structuring agents are generally removed by calcination, which consumes a lot of energy, or / and they are removed by extraction in organic solvents often toxic.

Gérardin et al (US2011 / 0028576 and WO2009 / 081000) describe nanostructured inorganic materials intended in particular for the preparation of catalysts. These materials are based on silicon and are obtained by means of nanostructuring agents consisting of micelles of diblock copolymers. But, if the nanostructured silica is not toxic, it is neither biodegradable, nor deformable, nor structured in thick layers as nanostructured polymers can be. Silica does not have the properties that would make it an ideal candidate for the transport of active molecules or for implantation in the body.

Berg et al (Berg MC, Zhei L., Cohen RE, Rubner MF, Biomacromolecules, 2006, 7, 357-364), Yang et al (Yang SY, JA Yang, Kim ES, Jeon G., Oh EJ, Choi KY, Hahn

S.K., Kim J.K., acsnano, 2010, 4, 3817-3822), Desai et al (D.A. Bernards, Desai T.A., Adv.

Mat, 2010, 22, 2358-2362; Bernards D.A., Desai T.A., Soft Matter, 2010, 6, 1621-1631) and

Martin et al (Martin F., Walczak R., Boiarski A., Cohen M., West T., Cosentino C, Ferrari M., J. of Controlled released, 2004, 102, 123-133) describe the nanostructuring of polymers. to form ultrathin porous materials in the form of membranes, their thickness not exceeding one hundred nanometers.

Mackay et al (Mackay M.E., Tuteja A., Duxbury M., Hawker C.J., Van Horn B.,

Guan Z., Chen G., Krishnan R.S., Science, 2006, 311, 1740-1743) describe the conditions necessary for the stability or segregation of polymer blends such as polystyrene and nanoparticles such as fullerenes. Their materials are not porous and are particularly intended for photovoltaic devices (Mackay et al, US2007 / 0212528).

Ye et al (Ye M., Kim S., Park K., J. of controlled released, 2010, 146, 241-260) disclose biopolymers, in the form of microspheres, for vectorizing proteins for therapeutic purposes. But the re-release profile is neither controlled nor continuous.

Johnson et al (Johnson S.A., Brigham E.S., Ollivier P.J., Mallouk T.E., Chem Mater,

1997, 9, 2448-2458) use zeolites as structuring agents for organic materials such as Bakelite. But the treatment methods are aggressive since they require pyrolysis and the use of hydrofluoric acid to release the porosity.

Wang et al (Wang XD, Tan B., Teng Q., Yao X., Su C, Wang W, Chem.

2010, 46, 970-972) describe organic nanoporous polymers consisting of

Trôger intended for heterogeneous catalysis applications.

Canelas et al (Canelas D.A., Herlihy K.R., DeSimone J.M., advanced review, 2009, 1,

391-404) describe studies relating to the use of nanoparticles formed of polymers and obtained by a "PRINT" method (Particle Replication In Nonwetting Templates) and intended for the vectorization of drugs and their release by enzymatic degradation of the container. .

One of the aims of the invention is to provide biodegradable nanostructured materials in the form of thick films, containing micelles having therapeutic properties, for medical applications.

Another object of the invention is to provide a process for the preparation of nanostructured biodegradable materials in the form of thick films, containing free pores, by gentle methods, without heating, without the use of aggressive treatments. Another object of the invention is to provide biodegradable nanostructured materials in the form of thick films, containing active molecules and allowing their release by single file effect, for therapeutic purposes. The invention relates to a method for preparing a nanostructured material containing free pores comprising the following steps:

A step of mixing an organic polymer in a solvent, and micelles to obtain a stable mixture of organic polymer and micelles,

A step of drying the stable mixture of organic polymer and micelles to remove the solvent and to obtain a nanostructured material containing micelles in the form of a dry film containing pores occupied by micelles,

A step of disassembling the micelles from the aforesaid film, in particular by washing in an aqueous medium, to obtain a nanostructured material in the form of a film containing free pores,

A step of drying the nanostructured material to remove the water and obtain a nanostructured material containing free pores,

said nanostructured material obtained being in the form of a dry film having a thickness of from 1 μιη to 500 μιη, in particular from 10 μιη to 200 μιη, in particular from 50 μιη to 150 μιη.

By "nanostructured material" is meant a material containing nano-objects whose size is from 1 nm to 100 nm. By "nanostructured material containing free pores" is meant a nanoporous material, characterized by the presence of interstices, qualified as "free pores" if these interstices are empty.

Among the nanostructured materials, there are therefore nanoporous materials, in which there are pores of nanometric size. The nanometric structure of the material of the invention gives it advantageous physical and chemical properties, which can be used in particular for the encapsulation of substances of interest and then their release for particularly therapeutic purposes. The pores can constitute cages or tubes, these materials are exploitable, among others, as host structure for molecules.

The materials according to the invention make use of "organic polymers" defined as macro molecules formed by repeating one or more unit (s) or monomer (s). These materials then have the advantage, in front of inorganic materials such as silica or zeolites, to be deformable and biodegradable, making them good candidates for biomedical applications, unlike inorganic materials. By "micelle" is meant an assembly consisting of molecules that tend to associate in solution. In the invention, the micelles constitute "jigs"; they allow nanostructuring by giving a nanometric size and a shape to the pores of said material. By "stable mixture" is meant a mixture formed from the organic polymer and micelles not evolving over time, the mixture being stable for at least one month.

By "disassembly" is meant the dissociative action of the structuring parts of the micelles. It frees empty spaces, free pores.

By "drying" is meant the step of removing any trace of solvent or water contained in the film.

By "dry film" is meant the form in which the material is obtained, its thickness being advantageously of the order of one hundred micrometers, which is about 1000 times greater than that of the materials described in the prior art. .

The thickness of the films obtained is one of the advantages of the present invention.

Another advantage of the invention is the preparation of nanostructured materials by gentle methods, without resorting to aggressive methods such as calcination or the use of organic solvents.

Another advantage is the simplicity of implementation of the preparation process for nanostructuring the material starting from the smallest scale to the material, bottom-up process, making the cost of production of the materials lower than the costs generated. by methods such as lithography, ion bombardment or layer-by-layer assembly.

Advantageously, the micelles are a complex of two structuring parts interconnected by one or more types of non-covalent reversible bonds.

The bonds allowing the two structuring parts constituting the micelles of the invention, to associate, are in particular electrostatic type bonds, which can be formed and discarded.

Particularly advantageously, the two structuring parts forming the micelles, consist of a double-hydrophilic block polymer (CBDH) containing a polyelectrolyte block and an oppositely charged polyelectrolyte, or being two double-hydrophilic block copolymers containing sequences. polyelectrolytes of opposite charges. By "polyelectrolyte" is meant an ionic polymer. The electrostatic type connections between the two structuring parts are thus allowed by electrical interaction between the parts and cationic anionics of said polyelectrolytes.

According to an advantageous mode of the process for preparing a nanostructured material, the micelles are lyophilized.

By "lyophilized micelles" is meant micelles isolated, dried, and retaining their properties. They can thus be stored for use in the process for preparing nanostructured materials.

Advantageously, the density of free pore is understood Ι, ^Ο χ ΙΟ ¹⁶ to I, 0x l0 ¹⁸ pores / cm ^3, in particular in the range of 2.0x 10 ¹⁶ 8.0x l0 to ¹⁶ pores / cm ^3.

The internal structure of the films was observed by transmission electron microscopy (TEM) on cryo-fractured film samples. The nanoscale structure was probed by atomic force microscopy (AFM).

Since the pores are in particular intended for the encapsulation of substances of interest for therapeutic purposes, the adjustment of the pore density may allow the control of the quantity of substance released for therapeutic purposes.

Advantageously, in this process of the invention for preparing a nanostructured material containing free pores, said pores have a size of from 1 nm to 200 nm, in particular from 2 nm to 100 nm, in particular from 5 nm to 20 nm.

The standard deviation of the lognormal distribution is about 0.3 for a pore diameter of about 9 nm.

One objective of the invention is to be able to adjust the pore size and adapt it to that of the molecules likely to be encapsulated therein. If the pores are occupied by compounds with therapeutic properties in order to vectorize them in the body in particular, the burst effect must be avoided and the unique file effect must be favored: the diffusion by the pores must prevail before the degradation of the polymer. However, the size of the pores according to the invention is adaptable to that of the compounds to be vectorized and released. For this, the pore size will not exceed twice the size of the diffusing compound. This point is particularly advantageous because the release of the active ingredient is then independent of the concentration of the compound to be released.

In an advantageous embodiment of the process for the preparation of the invention of a nanostructured material containing free pores, this comprises a step of mixing an organic polymer, in particular a biodegradable polymer, consisting of a homopolymer, a block copolymer or random copolymer, and micelles.

The materials according to the invention are of organic nature, biocompatible and biodegradable; they thus correspond to the definition of "biomaterals".

"Homopolymer" refers to a polymer derived from a single chemical species, all its reasons being therefore identical in nature.

In contrast to homopolymer, a "block copolymer" refers to a polymer containing several repeating units, at least two, therefore contains two different types of monomers. Within the polymer, each of the monomer types forms a block.

By "random copolymer" is meant a polymer containing several repeating units, at least two, thus containing two different types of monomers, the distribution of the different units being random.

In an advantageous embodiment of the preparation process of the invention, it comprises a step of mixing an organic polymer, in particular a biodegradable polymer, consisting of a homopolymer, and micelles,

said homopolymer being a polyester selected from poly-L-lactide, poly-D-lactide or poly-D, L-lactide.

Polyesters such as polylactide (PLA) have the advantage of being biocompatible and biodegradable by the action of esterases. On the other hand, PLA has the advantage of being able to be prepared with molar masses and very variable forms.

The PLA has the formula:

in which n represents the number of patterns.

PLA is obtained by ring opening and polymerization in the presence of a catalyst, lactide (lactic acid dimer). This is represented in the following diagram:

lactide PLA

Another advantage is to be able to use PLA in optically pure form or in the form of of racemic mixture. Indeed, lactic acid has two asymmetric carbons. It can therefore be used in chiral form (poly-L-lactide and poly-D-lactide), or in racemic form (poly-D, L-lactide). In another advantageous embodiment of the process of the invention, it comprises a step of mixing an organic polymer, in particular a biodegradable polymer, consisting of a copolymer consisting of two blocks, and micelles,

said blocks constituting the block copolymer being

A block of polylactide (PLA) in racemic form (poly-D, L-lactide, PDLLA) or in optically active dextrorotatory form (poly-D-lactide, PDLA) or levorotatory (poly-L-lactide, PLLA), and

The other block being chosen from polyethylene oxide (PEO) or polyglycolide.

The copolymers of the invention consist of two blocks, one necessarily being a block of PLA, chiral or racemic, the other block being a block of polyethylene oxide (PEO) or a block of polyglycolide (PGA).

The advantage of working with block copolymers is to be able to adjust the degradation time of said copolymer. In another advantageous embodiment of the process of the invention, it comprises a step of mixing an organic polymer, in particular a biodegradable polymer consisting of a random copolymer, and micelles.

said random copolymer being constituted

Polylactide (PLA) in racemic form (poly-D, L-lactide, PDLLA) or in optically active dextrorotatory form (poly-D-lactide, PDLA) or levorotatory (poly-L-lactide, PLLA), and

• and polyglycolide.

The random copolymers of the invention may consist of PLA, chiral or racemic, and polyglycolide (PGA).

The advantage of working with statistical copolymers is to be able to adjust the degradation time of said copolymer.

According to a particular embodiment of the preparation method of the invention, it comprises a step of mixing an organic polymer, in particular a biodegradable polymer, and micelles, said organic polymer being an organic homopolymer having a molar mass of from 3,000 to 1,000,000 g / mol,

in particular equal to 60,000 g / mol for the homopolymer of poly-L-lactide,

and in particular equal to 75,000 g / mol or 1,000,000 g / mol for the homopolymer of poly-DL-lactide.

The dispersion of nano-objects in the material requires a judicious choice between the size of said nano-objects and the radius of gyration of the polymer, the "radius of gyration" being related to the spatial extent occupied by the polymer. Thus, the miscibility between the micelles and the polymer depends on the molar mass of the latter.

When the radius of gyration is greater than the size of the micelles, the mixture of polymer and micelles is homogeneous, the range of gyration radii depending on the desired pore size.

As an illustration of this property, it has been calculated that the critical molar mass below which the 10 nm nano-object mixtures and the polymer are immiscible is 20,000 g / mol.

According to a particular embodiment of the preparation method of the invention, it comprises a step of mixing an organic copolymer, in particular a biodegradable copolymer consisting of two blocks, and micelles,

said copolymer having in particular the formula PEO-PDLLA, PEO-PDLA or PEO-PLLA, the blocks having for molar mass:

• 1,000 to 3,000 g / mol for the PEO block, in particular 1,880 g / mol,

5,000 to 500,000 g / mol for the PDLLA, PDLA or PLLA block, in particular 10,000 to 456,000 g / mol, and in particular 10,000, 77,322, 113,000 or 455,300 g / mol,

6,000 to 503,000 g / mol of total molar mass for the copolymer of formula PEO-

PDLLA, PEO-PDLA or PEO-PLLA, especially 11,880, 79,212, 114,880 or 457,180 g / mol.

The ring opening polymerization method of cyclic lactide dimers is used to synthesize PEO-PLA copolymers, the reaction being catalyzed by stannous octoate. In this method, the PEO block is terminated with a -OCH ₃ (MePEO-PLA) group.

The copolymers obtained are characterized by 1 H NMR.

The integration ratio of the -OCH ₃ group compared with the peak of the CH ₂ -CH ₂ -O group makes it possible to determine the molar mass of the PEO block. The resonance peaks corresponding to the groups -CH ₃ and -CH- make it possible to determine the polymerization index and the molecular mass of the PLA block. In the invention, the copolymers in which the PEO block has a molar mass of 1880 g / mol are advantageous because the PEO with a molar mass of 1880 g / mol is commercial.

According to a particular embodiment of the preparation method of the invention, it comprises a step of mixing a statistical organic copolymer, in particular a biodegradable copolymer, and micelles,

said copolymer being in particular poly-DL-lactide-polyglycolide, with a molar mass ranging from 20 000 to 2 000 000 g / mol, in particular equal to 1 000 000 g / mol, the fraction of poly-DL-lactide / polyglycolide being in particular equal to 50/50.

The molar mass of poly-DL-lactide-polyglycolide (PDLLA-PGA) must be at least equal to 20,000 g / mol, because for a lower molecular weight, the mixture is not homogeneous. For this molar mass in particular, the size of the nano-objects formed is of the order of 10 nm.

According to one embodiment of the process for preparing a nanostructured material of the invention, containing free pores, it comprises a step of mixing an organic polymer, in particular a biodegradable polymer, and micelles, which result from complexation. of two structuring parts with one or more types of non-covalent reversible bonds, in an aqueous solvent, said complexation being optionally followed by removal of the aqueous solvent, to give the micelles.

By "complexation" is meant the mode of association of the two structuring parts.

The expression "non-covalent reversible bonds" applies to electrostatic interactions existing between the two structuring parts of the micelles and explaining the formation of these micelles. These bonds are reversible because they can be formed and discard in response to a stimulus exerted by a parameter of the medium, for example the pH.

According to a particular embodiment of the process for preparing a nanostructured material of the invention, containing free pores, the complexation is followed by the removal of the aqueous solvent, and a step of lyophilization of the micelles, to obtain lyophilized micelles.

In an advantageous embodiment of the preparation process of the invention, the two structuring parts in an aqueous solvent are a double block copolymer. hydrophilic (CBDH) containing a polyelectrolyte block and an oppositely charged polyelectrolyte,

or are two double-hydrophilic block copolymers containing polyelectrolyte sequences of opposite charges,

the complexation having taken place in an aqueous medium at a pH ranging from 5.0 to 7.0, in particular ranging from 5.8 to 6.2, and especially equal to 5.5 or 6.0.

The charge of the polyelectrolytes of the invention depends on the pH value of the solution containing them. This is why mixing the two structuring parts must be done by checking the pH value and choosing it in the range of 5.0 to 7.0. This value will be set at 5.5 or 6.0.

The electrostatic force exerted between the groups of opposite charges carried by the two structuring parts is the motor of the formation of micelles, particularly sensitive to pH. According to an advantageous embodiment of the process for preparing a nanostructured material containing free pores, it comprises a step of mixing an organic polymer, in particular a biodegradable polymer, in an organic solvent, and micelles which result from a mixture of two structuring parts,

A structuring part being a hydrophilic double block copolymer (CBDH) made from polyethylene oxide PEO and from poly (methacrylic acid) PMAA and being represented by the formula EO "-b-MAA _m ,

in which

N denotes the number of ethylene oxide units, n varying in particular from 50 to

800

· M denotes the number of methacrylic acid units, m varying in particular from

10 to 100,

The ratio n / m being in particular between 3 and 10,

The other structuring part being an organic polyamine chosen from polyethyleneimine PEI, chitosan, polyvinylpyridine or polyallylamine, in particular PEI or chitosan,

said polyamine optionally having therapeutic properties, and being in particular chitosan, lidocaine, tetracaine, procaine, cimetidine, gentamicin, kanamycin, doxorubicin, gencitabine, or catecholamine, said micelles having the formula CBDH / polyamine, in particular EO "-b- MAA _m / polyamine wherein n and m have the values indicated above, and especially having the formulas EOi ₇₇ -b-MAA ₂₃ / PEI or EOi ₈ 2-b-MAA ₃₅ / PEI.

The term "hydrophilic double block copolymer (CBDH)" refers to a diblock copolymer, the two blocks of which, PEO and PMAA, are hydrophilic.

The CBDH according to the invention is designated "EO" -b-MAA _m ", the letter" b "meaning" block ", n and m denoting the number of units, respectively of ethylene oxide and methacrylic acid . At the time of formation of the micelles, the pH of the medium is from 5.0 to 7.0, in particular from 5.8 to 6.2, and especially equal to 5.5 or 6.0.

The formation of the micelles takes place at ambient temperature in an aqueous medium. After dissolution of the CBDH, a solution of polyamine is added in a pH medium defined above. After formation, they are optionally dialyzed to remove the excess of salts (if the polyamine is chitosan), then isolated or lyophilized to be optionally stored, or transferred after removal of the aqueous solvent in an organic medium compatible with the organic polymer before mixture of said organic polymer and said micelles.

If the micelles are used in the form of a lyophilizate for mixing with the organic polymer in solution in an organic solvent, then the lyophilizate is introduced beforehand into an organic solvent, identical to that of the polymer or different from it, subject to that it is compatible with that of the polymer.

- If the micelles have been prepared, freed from the water they contain without however being lyophilized, then they may be introduced into an organic solvent, identical to that of the polymer or different from the latter, provided that it is compatible with that of the polymer.

When the polyamine is polyethyleneimine (PEI), the pH value of the medium is advantageously maintained at about 6.0. When the polyamine is chitosan, the pH value of the medium is advantageously maintained at about 5.5. Under these pH conditions, the CBDH of the invention is polyanionic because of these acrylate groups, while the other structuring part of the micelles consisting of the polyamine is polycationic. The micelles are thus formed by electrostatic interaction. The micellization is optimal in this zone of pH.

The micelles are represented by the general formula "EO" -b-MAA _m / polyamine ".

The polyamine may be a potentially therapeutic molecule. Thus, the chitosan still named "oligochitosan", can be used for its antibacterial and antimicrobial properties, lidocaine, tetracaine, procaine, for their anesthetic properties, cimetidine for its anti-histamine properties, gentamicin and kanamycin for their antibiotic properties, doxorubicin and gencitabine for their anticancer properties, catecholamines as neurotransmitters, cardiocirculatory distress, vascular, cardiac or the nervous system.

In the process of the invention, a solution or a suspension of the aforementioned micelles is mixed with the block copolymer or random copolymer containing polylactide, previously designated. The mixture is in organic medium.

The n / m ratio is important for adjusting micelle size. According to a particular embodiment of the preparation method of the invention, it comprises a step of mixing an organic polymer, in particular a biodegradable polymer, and micelles of formula EO "-b-MAA _m / polyamine,

the ratio S = MAA / N representing the ratio between the number of carboxylate or carboxylic functions along the poly (methacrylic acid) PMAA chains and the number of amine functions, S being between 0.1 and 2, and being in particular equal to 1.

The micellization is optimal when S, the number of moles of anionic sites (COO) on the number of moles of cationic sites (NH ⁺ ), is close to 1.0.

According to a particular embodiment of the preparation process of the invention, it comprises a step of mixing an organic polymer, in particular a biodegradable polymer, and core / crown type micelles, with a dry diameter of from 1 nm to 200 nm, in particular from 2 nm to 100 nm, in particular from 5 nm to 20 nm.

To account for the effect of polydispersity of micelle sizes on scattered intensity, a log-normal distribution was used. The standard deviation of the log-normal distribution for micelles of about 9 nm dry diameter is approximately equal to 0.01.

From dynamic light scattering (DDL) measurements, the hydrodynamic diameter of the micelles was measured. But to know the actual and specific sizes of micelles, heart radius, crown thickness, the diffusion of small angle neutrons (DNPA) was used. It has been established that the size of the micelles strongly depends on the lengths of hydrophilic and polyelectrolytic blocks of the CBDHs forming the micelles. The heart of the micelles is spherical, dense and compact.

By way of illustration, the micelles of formula EO 1 ₇₇ -b-MAA ₂ 3 / PEI consist of PMAA chains, negatively charged and rendered insoluble by complexation with the amine, in particular with the PEI, positively charged. This heart is surrounded by a hydrophilic crown of PEO chains, swollen with the solvent. The change in length of the hydrophilic PEO block therefore causes a change in the thickness of the micelle crown. On the other hand, the stretching of the PEO chains depends on the nature of the solvent; they are more drawn in chloroform in particular than in water. Also, the dry diameter has been determined, in particular by MET, because it is directly related to the diameter of the pore.

Illustratively, for the micelles of formula EOI -b-MAA ₇₇ ₂ 3 / PEI, the size distribution histogram performed on 50 "objects" shows an average size of 9.2 nm with a narrow distribution. According to a particular embodiment of the preparation method of the invention, it comprises a step of mixing an organic polymer, in particular a biodegradable polymer, and micelles suspended in an organic solvent chosen from chloroform, chlorobenzene, dimethylsulfoxide or toluene,

in particular at the concentration of 0.05 to 0.2 g of organic polymer and micelles in 1 ml of solvent,

to obtain a stable mixture of organic polymer and micelles.

By "concentration" is meant the sum of the mass of organic polymer and the mass of micelles per ml of solvent.

For example, the mixture of organic polymer and micelles is stirred magnetically, in a common solvent, in particular anhydrous chloroform, at a speed of 300 rpm, at a temperature of 20 to 35 ° C., and especially at room temperature. room. It is stable for at least one month.

According to an advantageous embodiment of the preparation process of the invention, the micelles are dissolved in anhydrous chloroform, said solution then being mixed with the organic polymer, itself in solution in anhydrous chloroform.

According to an advantageous embodiment of the preparation process of the invention, it comprises a step of drying the organic polymer and micelle mixture, said drying step being carried out by evaporation of the organic solvent to obtain a dry film containing pores occupied by micelles, of thickness ranging from 1 μιη to 500 μιη, in particular from 10 μιη to 200 μιη, in particular from 50 μιη to 150 μιη.

The resulting film contains organic solvent molecules. The film may be left at room temperature, at atmospheric pressure, for a period of from 1 day to 7 days, in particular 2 days. The films are then, for example, placed under vacuum for 24 hours. hours to ensure complete evaporation of the organic solvent.

The resulting dry film contains micelles, serving as templates for the pores.

According to a particular embodiment of the process for the preparation of the invention of a nanostructured material containing free pores, this comprises a step of disassembling the micelles from the dry film,

said disassembly of the micelles being carried out in particular by immersion of the aforesaid dry film in an aqueous solution of pH> 8,

to obtain a nanostructured material in the form of a film containing free pores.

As seen above, the micelles used as structuring agents are formed via a reversible assembly induced by the application of a stimulus consisting of the pH. Thus, an inverse variation of this stimulus leads to the dissociation of the micelles and the solubilization of the copolymers. They can then be removed from the biopolymer by gentle washing. They thus serve as templates. The dried films can be immersed in an aqueous bath at pH> 8, for a few days, to ensure complete removal of the micelles from the PLA matrix. Traces of water are then removed.

In order to follow the exit state of the micelles of the copolymer matrix containing PLA, for example, the determination of primary amines with ninhydrin is carried out. The free ninhydrin solution, yellow, turns violet when it reacts with primary amines, especially PEI. Thanks to a calibration curve made beforehand, the optical density of the solution containing the amines makes it possible to determine the rate. This solution has therefore been studied by visible UV spectroscopy. Thus, the colorimetric assay by absorbance made it possible to follow the dissociation rate of the micellar complexes and the kinetics of exit of the parts coming from these. On the other hand, the dissociation of the micelles was verified by a fluorescence study: for example, the PEI was complexed with disodium fluorescein, the dissociation of the micellar complexes was then monitored by measuring the fluorescence of the aqueous solution washing, at defined time intervals.

Disassembly can therefore be done in particularly mild conditions, without consuming a lot of energy. In addition, the structuring agents can be recovered and recycled, resulting in an additional cost advantage for the production of nanostructured materials.

According to a particular embodiment of the process for the preparation of the invention of a nanostructured material containing free pores, this comprises a drying step, especially under vacuum, a nanostructured material in the form of a film containing free pores after disassembly of the micelles,

to remove the water molecules and obtain a nanostructured material containing free pores in dry film form.

The film is placed under vacuum at a temperature of 30 ° C or lower for 24 hours to obtain a dry film.

According to another advantageous mode, the process for the preparation of the invention of a nanostructured material containing free pores, this comprises the following steps:

A mixing step, in particular in chloroform,

An organic polymer being:

A homopolymer of polyester, in particular a poly-L-lactide, a poly-D-lactide or a poly-D, L-lactide,

said homopolymer having a molar mass of from 3,000 to 1,000,000 g / mol, especially equal to 60,000 g / mol for the homopolymer of poly-L-lactide,

and in particular equal to 75 000 g / mol or 1 000 000 g / mol for the homopolymer of poly-DL-lactide,

Or a block copolymer consisting of a polylactide block in racemic form (poly-D, L-lactide, PDLLA) or optically active dextrorotatory (poly-D-lactide, PDLA) or levorotatory (poly-L-lactide) , PLLA), and a block selected from polyethylene oxide PEO or polyglycolide (PGA),

said block copolymer in particular having the formula PEO-PDLLA, PEO-PDLA or PEO-PLLA, the blocks having for molar mass:

1,000 to 3,000 g / mol for the PEO block, in particular 1880 g / mol,

- 5,000 to 500,000 g / mol for the PDLLA, PDLA or PLLA block, in particular 10,000 to

456,000 g / mol, and especially 10,000, 77,332, 113,000 or 455,300 g / mol,

6,000 to 503,000 g / mol of total molar mass for the copolymer of formula PEO-PDLLA, PEO-PDLA or PEO-PLLA, in particular 11,880, 79,212, 114,880 or 457,180 g / mol,

Or a random copolymer consisting of polylactide in racemic form (poly-D, L-lactide, PDLLA) or optically active dextrorotatory (poly-D-lactide, PDLA) or levorotatory (poly-L-lactide, PLLA), and polyglycolide, especially poly-DL-lactide-polyglycolide (PDLLA-PGA),

said random copolymer being in particular poly-DL-lactide-polyglycolide with a molar mass of between 20,000 and 2,000,000 g / mol, in particular equal to 1,000,000 g / mol, the fraction of poly-DL-lactide / polyglycolide being in particular equal to 50/50,

► and micelles of formulas EOi77-b-MAA ₂ 3 / PEI with a ratio S = 1, the weight ratio of organic polymer / micelles being from 1 to 100, and in particular from 1 to 9,

to obtain a stable mixture of organic polymer and EOi ₇₇ -b-MAA ₂ 3 / PEI micelles,

■ a step of drying the stable mixture of organic polymer and EOI ₇₇ MAA ₂ -b- 3 / PEI, in particular by evaporation of the chloroform at room temperature and at atmospheric pressure, then in vacuo, and obtain a dry film containing pores occupied by micelles,

A step of disassembling the micelles from the aforesaid dry film by immersion of the dry film in an aqueous solution of pH> 8 to obtain a nanostructured material in the form of a film containing free pores,

A step of drying said nanostructured material to remove the water and obtain a nanostructured material containing free pores,

said material obtained being in the form of a dry film having a thickness of from 1 μιη to 500 μιη, in particular from 10 μιη to 200 μιη, in particular from 50 μιη to 150 μιη.

The organic polymer / micelles mass ratio is in particular between 1 and 9. In fact, it corresponds to organic polymer fractions / micelles. The fractions studied in particular are: 90% -10% (ratio equal to 9), 85% -15% (ratio equal to about 5.7), 80% -20% (ratio equal to 4), 70% -30% (ratio equal to 2.3), 60% -40% (ratio equal to 1.5) and 50% - 50% (ratio equal to 1), where the percentages are percentages by mass.

A mixture consisting of an organic polymer / micelles mass ratio of approximately 50% -50% may lose its homogeneity and exhibit phase separation. Therefore, it is preferable to work with a weight ratio organic polymer / micelles at least equal to 1.

The invention also relates to a process for preparing a nanostructured material containing micelles, comprising the following steps:

A step of mixing in a solvent of an organic polymer, in particular a biodegradable polymer, and of micelles to obtain a stable mixture of organic polymer and micelles,

A step of drying the stable mixture of organic polymer, in particular biodegradable, and micelles, to remove the solvent and obtain a nanostructured material containing micelles in the form of a dry film containing pores occupied by micelles, said material being obtained in the form of a dry film containing micelles with a thickness of from 1 μιη to 500 μιη, in particular from 10 μιη to 200 μιη, in particular from 50 μιη to 150 μιη.

These micelles can be in particular therapeutically interesting.

According to an advantageous embodiment, the method for preparing the invention of a nanostructured material containing micelles comprises a step of mixing in an organic solvent an organic polymer, in particular a biodegradable polymer, with micelles,

Said organic polymer being

1000 to 3000 g / mol for the PEO block, in particular 1880 g / mol,

° or a statistical copolymer, in particular biodegradable, consisting of polylactide in racemic form (poly-D, L-lactide, PDLLA) or in optically active dextrorotatory form (poly-D-lactide, PDLA) or levorotatory (poly-L-lactide, PLLA), and polyglycolide (PGA),

said random copolymer being in particular poly-DL-lactide-polyglycolide with a molar mass of between 20,000 and 2,000,000 g / mol, in particular equal to 1,000,000 g / mol, the fraction of poly-DL-lactide-polyglycolide being especially equal to 50/50, Said micelles being obtained by mixing two structuring parts in an aqueous solvent, the two structuring parts being a double-hydrophilic block copolymer containing a polyelectrolyte block and an oppositely charged polyelectrolyte,

or being two double-hydrophilic block copolymers containing polyelectrolyte sequences of opposite charges,

said aqueous solvent having a pH of from 5.0 to 7.0, in particular from 5.8 to 6.2, and especially of 5.5 or 6.0,

A structuring part being a hydrophilic double block copolymer (CBDH) made from polyethylene oxide (PEO) and from poly (methacrylic acid) PMAA and being represented by the formula EO "-b-MAA _m ,

in which

N denotes the number of ethylene oxide units, n varying in particular from 50 to 800, • m denotes the number of methacrylic acid units, m varying in particular from

10 to 100,

"The ratio n / m being in particular between 3 and 10,

The other structuring part being an organic polyamine chosen from polyethyleneimine PEI, chitosan, polyvinylpyridine or polyallylamine, in particular PEI or oligochitosan,

said polyamine optionally having therapeutic properties, and being in particular chitosan, lidocaine, tetracaine, procaine, cimetidine, gentamicin, kanamycin, doxorubicin, gencitabine, or catecholamine, said micelles having the formula CBDH / polyamine, in particular EO "-b- MAA _m / polyamine wherein n and m have the values indicated above, and in particular having the formulas EOi ₇₇ -b-MAA ₂₃ / PEI or EOi ₈ 2-b-MAA ₃₅ / PEI,

the polymer / micelles ratio being from 1 to 100, and in particular from 1 to 9, to obtain a stable mixture of polymer and micelles in an organic solvent.

According to an advantageous embodiment of the process for the preparation of the invention of a nanostructured material containing micelles,

the organic solvent used is chosen from chloroform, chlorobenzene, dimethylsulfoxide or toluene,

at the concentration of 0.05 to 0.2 g of organic polymer and micelles in 1 mL of solvent.

In another advantageous embodiment of the process for preparing the invention of a nanostructured material containing micelles, it comprises a step of drying a stable mixture of organic polymer, in particular biodegradable, and micelles,

said drying step being carried out in particular by evaporation of the organic solvent to obtain a dry film containing pores occupied by micelles,

and of thickness ranging from 1 μιη to 500 μιη, in particular from 10 μιη to 200 μιη, in particular from 50 μιη to 150 μιη.

The measurements indicate that there is not substantially any difference between the thickness of the films with and without micelles. According to another advantageous embodiment of the process for the preparation of the invention of a nanostructured material containing micelles, the micelles consist of a CBDH, in particular containing a polyelectrolyte block, in particular a polyanionic block, and an active ingredient containing an ionized group, in particular a cationic group, in particular an organic polyamine having therapeutic properties,

said polyamine constituting the active ingredient being chosen from chitosan, lidocaine, tetracaine, procaine, cimetidine, gentamicin, kanamycin, doxorubicin, gencitabine or a catecholamine.

The invention also relates to a method of encapsulation of one or more active principle (s) in a nanostructured material containing free pores defined above,

comprising a step of solubilizing the active ingredient (s) in a selective solvent, in particular an aqueous solvent, and a step of filtration under reduced pressure of the concentrated solution on the nanostructured material containing free pores,

the said active ingredient (s) encapsulated, used for its (their) properties in the pharmaceutical or cosmetic fields,

is (are) inorganic (s), in particular chosen from cisplatin, carboplatin, ruthenium and rhodium salts, gadolinium salts, iron oxides, complexes of gold, silver or copper,

or is (are) organic, synthetic or natural.

Encapsulation is performed by passing the solution containing the compound to be encapsulated onto the material, which acts as a filter.

The thickness of the nanostructured polymers according to the invention is of the order of one hundred microns, which is about 1000 times thicker than those described in the prior art. Thus, the nanostructured polymers of the invention, which allow a constant release rate, as mentioned above, also make it possible to encapsulate a larger quantity of compounds of therapeutic interest and thus to release them in a longer way in time. This is a particularly advantageous property when considering the implantation of said materials in the human body.

Among the encapsulable compounds, some organic have been previously mentioned, as well as their properties. But it is also possible to encapsulate inorganic compounds such as cisplatin and carboplatin (anticancer drugs), ruthenium and rhodium salts (anti-cancer potentials), gadolinium salts (useful in MRI imaging), oxides of iron (useful in the anticancer treatment by hyperthermia and MRI imaging), complexes of gold, silver or copper.

The invention relates to a nanostructured material in the form of a dry film containing free pores occupied by micelles as obtained by the implementation of the process for preparing a nanostructured material containing micelles and defined above.

The invention also relates to a nanostructured material containing free pores in dry film form as obtained by the implementation of the process for preparing a nanostructured material containing free pores and defined above.

The nanostructured material of the invention, containing free pores in the form of dry film can be used for the integration of an active ingredient for pharmaceutical use in said pores

chosen from among the substances

· Vitamins,

Anti-tumoral agents, in particular paclitaxel, alendronate, amonafide, leuprolide acetate, doxorubicin, gencitabine,

• anti-inflammatories, especially diclofenac, ibuprofen, ketoprofen,

• antibiotics, especially chitosan, gentamicin, kanamycin, · antifungals, especially ketoconazole,

• hormonal, including testosterone,

• anxiolytics, especially benzodiazepines,

• antidiabetic drugs, especially glicazide,

Antihypertensives, especially nifedipine; • antihistamines, including cimetidine,

• anesthetics, including lidocaine, tetracaine, procaine,

• neurotransmitters, especially catecholamines.

Advantageously, the active ingredients that can be encapsulated in the nanostructured materials according to the invention are intended for medical use and veterinary use.

The nanostructured material according to the invention, containing free pores in dry film form, can also be used for the integration of a substance of interest for cosmetic use in said pores,

chosen from among the substances

• anti-aging,

• Sun Creme,

• depigmenting,

• healing,

• moisturizing,

• fragrant.

The nanostructured material according to the invention, containing free pores in the form of a dry film, can also be used for the integration of an active ingredient in said pores, in order to protect against physical, chemical or chemical degradation. enzyme,

said active ingredient being in particular a protein.

The invention also relates to a stable mixture containing an organic polymer and micelles in an organic solvent,

Said organic polymer being

Or a block copolymer consisting of a polylactide block in racemic form (poly-D, L-lactide, PDLLA) or optically active dextrorotatory (poly-D-lactide, PDLA) or levorotatory (poly-L-lactide) , PLLA), and a block selected from PEO ethylene oxide or polyglycolide (PGA),

1000 to 3000 g / mol for the PEO block, in particular 1880 g / mol,

456,000 g / mol, and especially 10,000, 77,332, 113,000 or 455,300 g / mol,

Or a random copolymer consisting of polylactide in racemic form (poly-D, L-lactide, PDLLA) or optically active dextrorotatory (poly-D-lactide, PDLA) or levorotatory (poly-L-lactide, PLLA), and polyglycolide (PGA),

said random copolymer being in particular poly-DL-lactide-polyglycolide with a molar mass of between 20,000 and 2,000,000 g / mol, in particular equal to 1,000,000 g / mol, the fraction of poly-DL-lactide-polyglycolide being especially equal to 50/50,

Said micelles resulting from a combination of two structuring parts by one or more types of non-covalent reversible bonds, in an aqueous solvent having a pH of from 5.0 to 7.0, in particular ranging from 5.8 to 6.2, and in particular equal to 5.5; or 6.0,

A structuring part being a double-hydrophilic block copolymer containing a polyelectrolyte block and an oppositely charged polyelectrolyte,

especially a hydrophilic double block copolymer (CBDH) made from polyethylene oxide (PEO) and from poly (methacrylic acid) PMAA and being represented by the formula EO "-b-MAA _m ,

in which

N denotes the number of ethylene oxide units, n varying in particular from 50 to 800,

M denotes the number of methacrylic acid units, m varying in particular from 10 to

100

The ratio n / m being in particular between 3 and 10,

said polyamine optionally having therapeutic properties, and being especially chitosan, lidocaine, tetracaine, procaine, cimetidine, gentamicin, kanamycin, doxorubicin, gencitabine, or catecholamine, said micelles having the formula CBDH / polyamine, in particular EO "-b- MAA _m / polyamine wherein n and m have the values designated above , and especially having the formulas EOi ₇₇ -b-MAA ₂₃ / PEI or EOi ₈ 2-b-MAA ₃₅ / PEI,

the polymer / micelle ratio being from 1 to 100, and especially from 1 to 9.

FIGURES FIG. 1 represents the ionization domains of the PMAA blocks and the polyamine blocks according to the pH, as well as the micellization domain of the micelles of formula CBDH / polyamine, in particular micelles of the formulas EOi ₇₇ -b-MAA ₂ 3 / PEI and EOis ₂ -b- MAA ₃₅ / PEI.

Figures 2a and 2b show the variation of the hydrodynamic diameter (·) and the diffused intensity (■) of micelles based on PEO-PMAA and PEI, and based on PEO-PMAA and oligochitosan (a and b respectively) as a function of pH with S = 1.

Figure 3 shows the variation in hydrodynamic diameter (·) and scattered intensity

(■) micelles of PEO177-PMAA23 / PEI as a function of the molar ratio S at pH = 6.

FIG. 4a shows the scattered intensity as a function of the wave vector by EO177-MAA23 / PEI complex micelles in D ₂ 0 at a concentration of 0.03 g / mL; (o): experimental curve, (-): heart-crown model.

Figure 4b shows the intensity diffused by EO177-MAA23 / PEI complex micelles in CDCl ₃ at a concentration of 0.005 g / mL; (o): experimental curve, (-): heart-crown model.

FIG. 4c represents the intensity diffused by a mixture of MePEO-PLA (114 880 g / mol) and EO177-MAA23 / PEI complex micelles (70% and 30% respectively) in CDCl ₃ at a concentration of 0.16 g / ml. The appearance of the peak structure shows that the medium is nanostructured. (o): experimental curve (-): model including the structure factor.

FIG. 5 represents the transmission electron microscopy (TEM) image of E0 ₁₇₇ -MAA ₂₃ / PEI micelles. Scale bar = 50 nm. The size distribution histogram is performed on 50 objects. The average size is 9.2 nm.

FIG. 6 is a transmission electron microscopy (TEM) replica of a cryofractured film prepared with MePEO-PLA (114 880 g / mol) and complex micelles EO177-MAA23 / PEI (70%) and 30% respectively ) then washed at pH>8; scale bar = 100 nm. The arrow shows a boundary between two different orientation zones of the fracture surface with respect to the metallization direction. Histogram of size distribution indicating an average size of 8 ± 2 nm.

Figure 7, on the left, is the AFM (Ιμιη ^χ ΐμιη) snapshot showing the nanostructures in a MePEO-PLA biopolymer matrix (114 880 g / mol) and EOiw-MAA ₂₃ / PEI complex micelles (70% and 30%). % respectively). On the right is the AFM (Ιμΐϊΐ ^χ ΐμπι) image showing the nanoporous structure in the same matrix after extraction of the complex micelles by washing at pH = 8. EXAMPLES

Meaning of the abbreviations used:

CHCI ₃ : chloroform

CDCI ₃ : deuterated chloroform

1H NMR: Nuclear Magnetic Resonance of the proton

δ: displacement of the proton, expressed in ppm

DMSO: dimethylsulfoxide

M ": nominal mass

MET: Transmission Electron Microscopy

NaOH: sodium hydroxide

DDL: Dynamic Diffusion of Light

DNPA: Neutrons scattering at Les Petits Angles

AFM: Atomic Force Microscopy Features / types of analysis devices used:

1H NMR: Brucker NMR spectrometer, 600 MHz. These measurements were carried out at the Institute of Topology and Systems Dynamics (ITODYS), CNRS -Université Paris Diderot.

DDL: digital Brookhaven BI9000AT

DNPA: PAXY spectrometer at the Léon Brillouin laboratory at CEA Saclay.

cryofracture: Institute of Physics and Chemistry of Materials of Strasbourg (CNRS-University of Strasbourg).

MET: Tecnai 12 Spirit FEI microscope at accelerating voltage of 120 kV; micrographs collected using an ORIUS 832 GATAN camera at the Chemistry of Condensed Matter Chemistry Laboratory (CNRS -Université Pierre and Marie Curie / Collège de France). AFM: These measurements were carried out at the CNRS-Université d'Orléans Divisional Research Center on Divided Matter using a Nanoscope III (Digital Instruments Corp.)

Varian UV-Visible Spectrophotometer, Cary ® 50

fluorescence spectrophotometer: Cary Eclipse, Varian

pH meter: Mettler Toledo SevenMulti, equipped with a microelectrode.

Reagents and solvents used

Dimers and homopolymers L and D, L-lactide (Mn = 60,000 and 75,000 g / mol), monoethylether polyethylene oxide (MePEO, Mn ~ 2,000 g / mol), stannous octoate, polyethyleneimine ( PEI, Mn ~ 600 g / mol), oligochitosan (or chitosan) (Mn ~ 5000 g / mol), the solution of ninhydrin (2% in DMSO), fluorescein sodium (C ₂ oHi ₀ Na ₂ 05) dichloromethane (CH ₂ Cl ₂ ), chloroform (CHCl ₃ ), toluene, diethyl ether, d-chloroform (CDCl ₃ , Cambridge Isotope) and D ₂ O were purchased from Sigma-Aldrich (France) and used as is unless specified later. The hydrophilic double block copolymers (CBDH) polyethylene oxide - poly (methacrylate acid) (PEO - PMAA), of different molecular weights, were purchased from Polymer Source (Canada).

Example 1 Synthesis of Lactide Copolymers MePEO-PLA

After dissolving the lactide (L or D, L) in a minimum volume of anhydrous ethyl acetate under heat (to ensure complete solubilization), the solution was stirred magnetically for 24 hours. The reagent was recrystallized by filtration of the solution on nylon filters of porosity 0.45 μιη (Whatman). The crystals were then placed in the oven at 40 ° C under vacuum to evaporate the solvent residues.

The stannous octoate was previously diluted in a solution of anhydrous toluene at a concentration of about 0.15 g / mL.

The ring opening polymerization method of cyclic lactide dimers was used to synthesize MePEO-PDLLA copolymers (Al Helou, N. Anjum, MA Guedeau-Boudeville, M. Rosticher, A. Mourchid, Polymer, 2010). 51, 5440-5447, SK Agrawal, N. Sanabria-DeLong, GNTew, SRBhatia, Langmuir, 2008, 24, 13148-13154). MePEO was used to initiate polymerization through the opening of the dimeric ring by the single free hydroxyl group of the MePEO chain.

For this, the MePEO was dissolved in anhydrous toluene in a three-necked flask, placed in an oil bath at 130 ° C. under a stream of nitrogen and with continuous stirring. To dry the PEO, the balloon was topped with a Dean-Stark fixture. The water-toluene azeotrope (boiling point 84 ° C) condensed first with the refrigerant. Traces of water were thus removed from the toluene solution. The recrystallized dimers of lactide were then introduced into the flask and stirred until complete dissolution. A second volume of toluene was removed from the Dean-Stark and the system was refluxed under nitrogen until a new volume of toluene was condensed in the decanter. The stannous octoate was then added using a syringe to catalyze the reaction. The mixture was left under mechanical stirring at 130 ° C for 24 hours under positive nitrogen pressure. The reaction was stopped by cooling the solution to room temperature. The product obtained was dissolved in a minimum volume of CHCl ₃ with stirring. The polymer was then precipitated by pouring the chloroform solution dropwise into a solution of diethyl ether with stirring. The precipitated product was then filtered through a nylon filter with a porosity of 0.45 μιη. This last precipitation and filtration step was repeated several times to ensure precipitation and complete recovery of the polymer. Finally, the recovered product was evacuated at room temperature for 2 days for complete evaporation of the solvents. The yield of the syntheses varied from 60 to 90% depending on the molecular molar masses of the final polymer.

Example 2: Characterization of MePEO-PLA copolymers by ¹ H NMR

The analyzes of the results of the experiments on the products were carried out by determining exactly the mean molecular weight of the MePEO (nominal mass Mn = 2000 g / mol) thanks to the resonance peak of the OCH ₃ group located at 3.4 ppm. Comparing the integration ratio of this specific peak with the integrated value of the peak CH ₂ -CH ₂ -0 (δ = 3.7 ppm;

4 xn protons), the actual molecular weight of the MePEO used was calculated, Mn = 1880 g / mol. The polymerization index and the molecular weight of the PL A blocks were determined from the resonance peaks corresponding to the CH ₃ (3 xy protons) and CH (1 xy protons) groups. Table 1 shows the results of the NMR experiments made on the polymers that we used.

table 1

Average molecular masses and characteristics of synthesized polymers. Example 3 Preparation of micelle suspensions composed of CBDH PEO-PMAA and a polyamine

Different solutions were prepared in deionized water (MilliQ, Millipore, 18 ΜΩ ηι ¹ , France). To prepare suspensions of PEO-PMAA / polyamine complex micelles, a solution of the PEO-PMAA copolymer and a stock solution of the polyamine are mixed under certain conditions. The ratio S, defined as the number of carboxylate and carboxylic functions along the PMAA chains per amino function, S = MAA / N, is very important and influences the characteristics of the suspensions.

Typically, the preparation of a suspension of complex micelles from a CBDH EOi ₇₇ -MAA ₂ 3 (177 and 23 indicating the degree of polymerization of the chains PEO and PMAA respectively) and of polyamine for a ratio S = 1, a was carried out as follows:

50 mg of EO 1 ₇₇ -MAA ₂ 3 copolymer were directly weighed into a glass tube, thoroughly cleaned and dried, and 5 ml of deionized water was added to obtain complete dissolution of the copolymer. 1.2 ml of a mother solution of PEI at 0.1 M in amine functions was then added and the mixture was stirred magnetically for 2 hours, the pH value of the mixture being approximately equal to 6. With PEI, the final pH n need not be adjusted.

In contrast, the preparation of micelle suspensions containing the oligochitosan required the adjustment of the pH to the value of 5.5, by addition of a solution of 0.1 M NaOH, in the two stock solutions, as well as the adjustment of the pH value of the final suspension. The resulting suspension was then dialyzed against an aqueous solution at pH = 5.5 for one week and the excess salts were removed by measuring the conductivity of the dialysis water after each water change. The dialysis operation was necessary in the case of oligochitosan because it contains salt, which is not the case of PEI.

At the end of the complexation procedure, the suspensions were lyophilized and the recovered products were stored at 4 ° C for later use.

On the other hand, the formation of polyionic complexes as a function of the pH of the solution has been studied and described in the following example.

Example 4 Study of a PEO-PMAA / Polyamine System as a Function of the pH

The pH range on which the micelles were formed and defined was determined by dynamic light scattering (DDL).

FIG. 1 corresponds to the diagram of the ionization domains of the PMAA and polyamine blocks as well as the domain of micellization.

The electrostatic interactions occurring between the PMAA blocks and the polyamine blocks induce the formation of the micellar complexes. The pKa values of the various polyelectrolytes studied make it possible to predict the existence of a pH range for which both the PMAA blocks and the polyamine blocks are ionized. This range corresponds to pH values between 4.5 and 8 approximately. To better underline the effects of pH on the structure and formation of micelles, the mixtures were studied by DDL. For this purpose, a stock solution of EO 1 ₇₇ -MAA ₂ 3 / polyamine with a ratio S = 1 was prepared and then divided into 11 solutions of equal volumes. Then, the pH value of each of the daughter solutions was adjusted to a different value (the mother solution was divided into several solutions to avoid the unnecessary addition of salts) and the evolution of the hydrodynamic diameter, Ζ¾, was studied by DDL.

It can be seen in FIGS. 2a and 2b that the curves representing the hydrodynamic diameter Ζ¾ and the diffused intensity mark a sudden variation when the pH approaches the value of 5. The pH scale can be divided into two parts:

pH <5 and pH> 7: The intensity values measured are small and of the same order of magnitude for PEI and oligochitosan. The measured hydrodynamic radii are weak with high polydispersity. The objects present in the solution are not very diffusing. They are formed by unaggregated and very low density polymer chains;

<PH <7: The intensity values measured are high. The measured hydrodynamic radii are of the order of 44 nm for PEI and 53 nm for oligochitosan. In both cases, the polydispersity indices are low and the suspension is monodisperse with compact scattering objects.

The curves of FIG. 2 make it possible to assert that the optimum conditions for the formation of well-aggregated and monodisperse micelles correspond to pH values such that <pH <7. The pH value of the micellization solution has therefore been fixed. to 6 unless otherwise stated.

The effect of the molar ratio S, another important parameter affecting the formation and the size of the micelles, is identical for the micelles, that they contain the PEI or the oligochitosan.

Results for EOi ₇₇ -MAA ₂ 3 / PEI complex micelles were presented later. When the molar ratio S increases by 0.2, the majority population of diffusing objects moves slightly towards the increasing values of the hydrodynamic diameter and it becomes more and more narrow with the increase of S. In Figure 3, the evolution of the average size has been represented. The average hydrodynamic diameter as well as the intensity of scattered light increase depending on the value of the ratio S.

This result shows that even for a molar ratio of 0.2, the anionic block of PMAA still reacts with the cation block of PEI to form micelles with smaller hydrodynamic diameters and controlled by the PEO ring that surrounds and stabilizes the formed core by polyelectrolytes.

Additional confirmation was provided by the study of micelle morphology by small angle neutron scattering (DNPA). Figure 4a shows the intensity diffused by a micellar solution in deuterated water. The shape of the curve is characteristic of diffusion by nanometric objects. The use of the core-crown model makes it possible to account for the experimental measurements and to deduce the core radius, which is 4.4 nm, the extension of the corona (approximately 4 nm in water) and the aggregation number. = 28. These measurements are in good agreement with the direct observation of dried micelles, made by transmission electron microscopy, which gives an average size of 9.2 nm with a narrow distribution as can be seen in Figure 5.

In the following, the micelles used were prepared at a pH = 6 and for a molar ratio S = 1.

Example 5 Preparation of Nanostructured MePEO-PLA Copolymer Films

The method described here was used for the preparation of all nanostructured samples in anhydrous chloroform chosen as a common solvent for MePEO-PLA micelles and copolymers. Typically, to prepare a film of copolymers and micellar complexes contained in the film, with mass ratios of 70% and 30% for the copolymer and the micelles respectively, it was proceeded as follows:

micelle mixture and copolymer: 30 mg lyophilized micelles were dissolved in 1 ml of chloroform, the solution being turbid at first. The organic solution was then vortexed and then placed in an ultrasonic bath for a few minutes, causing the disappearance of turbidity. 70 mg of PLA-based copolymer were added, the dispersion of the micelles being again ensured by means of an ultrasound bath. The organic solution was then deposited on glass slides using a pipette. After evaporation of the chloroform at room temperature for 48 hours, then under vacuum for 24 hours, the dry films were obtained.

During this step, the DNPA was used to verify that the solvent permutation does not induce a change in the morphological properties of the micelles. The Figure 4b shows that the intensity diffused by the micelles is well adjusted by the heart-crown model. The study of the PLA-based copolymer mixture and complex micelles in deuterated chloroform gives rise to a localized correlation peak at q = 0.019 Å ^-1 as can be seen in Figure 4c, which shows that the mixture is structured on the basis of nanoscale.

disassembly: The films obtained were then immersed in an aqueous solution at pH> 8 to initiate the decomplexation of the micelles and promote their elimination. The films were thus left in the aqueous bath for a few days, to ensure the complete removal of the micelles from the PLA-based copolymer matrix and then re-evacuated for 24 hours to evaporate the traces of water.

The micelle exit state of the copolymer matrix was monitored by determination of primary amines with ninhydrin (S. Prochazkova, K. M. Varum, K. Ostgaard, Carbohydrate Polymers, 1999, 38, 115-122).

The method used is described below.

To a 1 mL sample of an aqueous solution containing amino functions, 1 mL of a ninhydrin solution (diluted to 2% in DMSO) was added. The mixture was then shaken by hand briefly and then placed in a boiling water bath for 30 minutes to initiate the reaction between the ninhydrin and the amino groups. After cooling the solution, 5 mL of water / ethanol (50/50 v / v) was added and the mixture was vortexed for 15 seconds to oxidize the excess ninhydrin. The absorbance of the solution was then measured, in visible light, the zero absorbance being performed using a sample of pure water treated in the same manner as above. As further evidence of PEI output kinetics observed by the ninhydrin assay method, PEI was labeled with a fluorescent molecule, fluorescein sodium. For this, a solution of PEI at a concentration of 0.1 M in the amine function was prepared. The sodium fluorescein was added to the PEI solution, shaken for 24 hours to ensure electrostatic complexation between the negatively charged fluorescein in aqueous solution and the PEI polycations, positively charged at the working pH (neutral pH). A ratio of 0.03% by number of moles of fluorescein to PEI was used to avoid the phenomenon of "quenching". Finally, the labeled PEI solution was used for the manufacture of micelles according to exactly the same procedure as previously described. The addition of negative electrostatic charges provided by fluorescein in the complexes and the calculation of the molar ratio S is negligible since they represent only a fraction of 0.0003. A typical example of a film washed with water, studied by transmission electron microscopy, is shown in FIG. 6. It should be remembered that the fracture is not necessarily flat. Areas of different levels and orientations are visible relative to the direction of deposition. This is clearly highlighted by an arrow in Figure 6 where a large, continuous, dark area is followed by a lighter area characteristic of a step. A statistic on the size of 180 identifiable objects on the plate that has characteristic shading of nanoscale holes in the fracture surface was made to determine the average pore size. The average size obtained for this plate is 8 nm, with an uncertainty of ± 2 nm due to the thickness of the deposited metal layer. Measurements obtained from other microscopy micrographs made it possible to deduce nanopore sizes between 8 and 12 nm ± 2 nm, in good agreement with the diameter of the micelles E0 ₁₇₇ - MAA ₂₃ / PEI used to induce the nanoporosity.

The nanostructuration of polylactide films using micelles and the creation of porosity by washing with water at pH> 8 were also studied by near-field microscopy. A typical example obtained for the nanostructuration of MePEO-PLA (114 880 g / mol) at the fraction of 70%, by micelles EO177-MAA ₂₃ / PEI at the fraction of 30%, is shown in Figure 7, left. In this case, a better distribution is observed for the emerging particles spread over the entire surface of the film. The films were washed with an aqueous solution at pH = 8 to verify the nature of the objects observed. The observations discussed above are clearly highlighted in the right-hand picture of Figure 7. The surface of the film is homogeneous after washing and the pores are well distributed. It should be noted that the sizes measured with the AFM are generally exaggerated because of the convolutions between the tip of the probe and the observed object. Example 6 Preparation of nanostructured PLA polymer films, with or without polyglycolide

The method described here was used for the preparation of all the nanostructured samples, in anhydrous chloroform chosen as a solvent common to micelles and PLA polymers, copolymerized or not with the polyglycolide. Typically, to prepare a film of polymers and micellar complexes contained in the film, with mass ratios of 70%> and 30%> for the polymer and the micelles respectively, it was proceeded as follows:

micelle mixture and polymer: 30 mg lyophilized micelles were dissolved in 1 mL of chloroform, the solution being turbid at first. The organic solution was then vortexed and then placed in an ultrasonic bath for a few minutes, causing the disappearance of turbidity. 70 mg of PLA-based polymer were added, the dispersion of the micelles being again ensured by means of an ultrasound bath. The organic solution was then deposited on glass slides using a pipette. After evaporation of the chloroform at room temperature for 48 hours, then under vacuum for 24 hours, the dry films were obtained.

disassembly: The films obtained were then immersed in an aqueous solution at pH> 8 to initiate the decomplexation of the micelles and promote their elimination. The films were thus left in the aqueous bath for a few days, to ensure the complete removal of the micelles from the PLA-based polymer matrix and then re-evacuated for 24 hours to evaporate the traces of water.

The micelle exit state of the polymer matrix was monitored by determination of primary amines with ninhydrin (Prochazkova, KM Varum, K. Ostgaard, Carbohydrate Polymers, 1999, 38, 115-122) and by monitoring the fluorescent molecule output, fluorescein sodium, previously attached to the amine.

EXAMPLE 7 Analysis of the MePEO-PLA Copolymer Samples by Nuclear Magnetic Resonance of the Proton ( ¹ H NMR)

1 H NMR was used in this study to determine the structure of synthetic products of copolymers containing polylactide. The polymers to be analyzed were dissolved in deuterated chloroform at room temperature at a concentration of about 15 mg / g. The chemical shift was calibrated against that of the solvent peak. Example 8 Dynamic Light Diffusion Analysis (DDL)

To perform these measurements, a light scattering assembly equipped with a helium-neon laser emitting at a wavelength of 632 nm and equipped with a goniometer, was used. The correlation function was obtained using a Brookhaven BI9000AT digital self-correlator. The samples were filtered on syringe filters, aqueous or organic, with a porosity of 0.45 μιη and 0.2 μιη in order to eliminate any trace of pollution. The correlation function obtained made it possible to determine the hydrodynamic size in the high dilution regime. Example 9: Diffusion Analysis of Neutrons at Small Angles (DNPA)

The DNPA experiments were carried out on the PAXY spectrometer at the Léon Brillouin laboratory at CEA Saclay, this diffusion assembly being equipped with a two-dimensional detector. Various spectrometer configurations were used to cover an area of wave vector q including 0.003 0.4 TO ^"1.

The polymer solutions were prepared in deuterated solvents, water and chloroform, to enhance the contrast between the polymers and the solvent and at the same time reduce the inconsistent signal caused by the hydrogen atoms of the sample. Dry films have been studied between two quartz windows.

Example 10: Transmission Electron Microscopy (TEM) Analysis

The dried films were studied by Cryo fracture. For this, the films were fixed between two specific copper supports moistened with a drop of chloroform to ensure good adhesion between the sample and its support and the whole was immersed in a bath of nitrogen liquid to ensure rapid freezing of the sample. The supports were then abruptly separated by a mechanical signal thus causing the longitudinal fracture of the film. A thin layer of platinum was evaporated obliquely on both sides of the fractured film to form the replica, and then a carbon layer was evaporated perpendicular to the samples to fix the platinum replica. At the end of this step, the samples, with the replicas, were recovered and immersed in a chloroform bath to remove the sample residues and thus wash the platinum replicas. The replicas were then recovered on microscopic grids and observed by the MET. The replicas of the samples were examined using a Tecnai 12 Spirit FEI microscope at an accelerating voltage of 120 kV, and the micrographs collected using a camera.

Example 11 Analysis by Atomic Force Microscopy (AFM)

AFM in dynamic mode was used because this mode does not cause degradation of the surface of the samples and is thus suitable for the study of flexible samples, including organic polymer films. The organic films of copolymers based on polylactide, nanostructured or not, before and after washing at pH> 8, were studied. Example 12: Determination of primary amines with ninhydrin followed by ultraviolet-visible spectroscopy and fluorescence spectroscopy

The absorbance of the samples is measured with a Varian UV-Visible spectrophotometer, Cary ® 50. The samples were studied in quartz tanks with an optical path of 2 mm thick, 10 mm wide and 30 mm high. .

Fluorescence spectroscopy measurements were made on a Cary Eclipse fluorescence spectrophotometer, Varian, in quartz tanks having an optical path 2 mm thick, 10 mm wide and 30 mm high. Example 13: Measurements by pH-metrics

The pHmetry was used to follow the determination of the carboxylic acid functions present on the PMAA blocks of the PEO-PMAA copolymers. The degree of polymerization and the pKa were thus determined. Aqueous solutions of PEO-PMAA copolymers at 4 mg / ml were determined by the regular addition of measured volumes of a 0.1 M sodium hydroxide solution. The oligochitosan titration in its lactate salt form was carried out by the addition of a 0.5M sodium hydroxide solution in the oligochitosan solution. The PEI titration was carried out by adding a 0.5M hydrochloric acid solution in the PEI solution.

Example 14 Dialysis

The aqueous dialyser solutions were introduced into dialysis membranes immersed in a deionized water reservoir whose pH was adjusted to 6, the pH of the dialysis water being set at the same pH as that of the solutions of the complexes. The output of the salt was monitored by measuring the conductivity of the reservoir water and thus this water was changed several times a day until the measured conductivity remained at a constant value. The membranes, based on regenerated cellulose (SpectraPor), form entangled networks thus generating a cutoff threshold. This threshold (expressed in Daltons) was chosen as a function of the molecular weight (in g / mol) of the samples to be purified: for diblock copolymers with a molecular mass of 9,800 g / mol, a cut-off threshold of 2000 Daltons was chosen and for diblock copolymers with a molar mass of 17,000 g / mol, this threshold was chosen at 6,000-8,000 Daltons.

Claims

1. Process for preparing a nanostructured material containing free pores comprising the following steps:

■ a step of mixing an organic polymer in a solvent, and micelles to obtain a stable mixture of organic polymer and micelles,

■ a step of drying the stable mixture of organic polymer and micelles to eliminate the solvent and obtain a nanostructured material containing micelles in the form of a dry film containing pores occupied by micelles,

■ a step of disassembling the micelles from the above-mentioned film, in particular by washing in an aqueous medium, to obtain a nanostructured material in the form of a film containing free pores,

■ a step of drying the nanostructured material to eliminate the water and obtain a nanostructured material containing free pores,

2. Process for preparing a nanostructured material according to claim 1, in which the micelles are a complex of two structuring parts linked together by one or more types of reversible non-covalent bonds, in particular the micelles being made up of a double-hydrophilic block polymer (CBDH) containing a polyelectrolyte sequence and a polyelectrolyte of opposite charge, or being two double-hydrophilic block copolymers containing polyelectrolyte sequences of opposite charges, the micelles being in particular lyophilized.

3. Process for preparing a nanostructured material containing free pores according to one of claims 1 or 2,

in which the density of the pores is comprised from 1.0>< 10 ¹⁶ to 1.0>< 10 ¹⁸ pores/cm ³ , in particular comprised from 2.0x 10 ¹⁶ to 8.0x 10 ¹⁶ pores/cm ³ and in particular in which the free pores have a size ranging from 1 nm to 200 nm, in particular from 2 nm to 100 nm, in particular from 5 nm to 20 nm.

4. Process for preparing a nanostructured material containing free pores according to one of claims 1 to 3, comprising a step of mixing an organic polymer, in particular biodegradable, consisting of a homopolymer, a block copolymer or a statistical copolymer, and micelles,

said homopolymer being in particular a polyester chosen from a poly-L-lactide, a poly-D-lactide or a poly-D,L-lactide,

said copolymer being in particular made up of two blocks,

• a block of polylactide (PLA) in racemic form (poly-D,L-lactide, PDLLA) or in optically active dextrorotatory form (poly-D-lactide, PDLA) or levorotatory form

(poly-L-lactide, PLLA), and

• the other block being chosen from polyethylene oxide (PEO) or polyglycolide,

said copolymer being in particular a random copolymer consisting

“polylactide (PLA) in racemic form (poly-D,L-lactide, PDLLA) or in optically active dextrorotatory (poly-D-lactide, PDLA) or levorotatory (poly-L-lactide, PLLA) form, and

• and polyglycolide,

the molar mass of said homopolymer being in particular between 3,000 and 1,000,000 g/mol, in particular equal to 60,000 g/mol for the poly-L-lactide homopolymer,

and in particular equal to 75,000 g/mol or 1,000,000 g/mol for the poly-DL-lactide homopolymer,

said copolymer having in particular the formula PEO-PDLLA, PEO-PDLA or PEO-PLLA, the blocks having in particular the molar mass:

· 1,000 to 3,000 g/mol for the PEO block, in particular 1,880 g/mol,

• 5,000 to 500,000 g/mol for the PDLLA, PDLA or PLLA block, in particular 10,000 to 456,000 g/mol, and in particular 10,000, 77,332, 113,000 or 455,300 g/mol,

• 6,000 to 503,000 g/mol of total molar mass for the copolymer of formula PEO-PDLLA, PEO-PDLA or PEO-PLLA, in particular 11,880, 79,212, 114,880 or 457,180 g/mol, said copolymer being in particular poly-DL-lactide-polyglycolide, with a molar mass of from 20,000 to 2,000,000 g/mol, in particular equal to 1,000,000 g/mol, the fraction of poly-DL-lactide/polyglycolide being in particular equal to 50/ 50.

5. Process for preparing a nanostructured material containing free pores according to one of claims 1 to 4, comprising a step of mixing an organic polymer, in particular biodegradable, and micelles, which result from the complexation of two structuring parts by one or more types of reversible non-covalent bonds, in a aqueous solvent, said complexation possibly being followed by elimination of the aqueous solvent, to give the micelles,

and optionally comprising a step of freeze-drying the micelles, to obtain freeze-dried micelles,

the two structuring parts in an aqueous solvent advantageously being a double-hydrophilic block copolymer containing a polyelectrolyte sequence and a polyelectrolyte of opposite charge,

the complexation having advantageously taken place in an aqueous medium at a pH of 5.0 to 7.0, in particular of 5.8 to 6.2, and in particular equal to 5.5 or 6.0.

6. Process for preparing a nanostructured material containing free pores according to claim 5, comprising a step of mixing an organic polymer, in particular biodegradable, in an organic solvent, and micelles, which result from a mixture of two structuring parts,

■ a structuring part being a hydrophilic double block copolymer

(CBDH) consisting of polyethylene oxide PEO and poly(methacrylic acid) PMAA and being represented by the formula EO„-b-MAA _m ,

in which

• n designates the number of ethylene oxide units, n varying in particular from 50 to 800,

• m designates the number of methacrylic acid units, m varying in particular from 10 to 100,

• the n/m ratio being in particular between 3 and 10,

■ the other structuring part being an organic polyamine chosen from polyethyleneimine PEI, chitosan, polyvinylpyridine or polyallylamine, in particular PEI or chitosan,

said polyamine possibly possessing therapeutic properties, and being in particular chitosan, lidocaine, tetracaine, procaine, cimetidine, gentamicin, kanamycin, doxorubicin, gencitabine, or a catecholamine, said micelles having the formula CBDH/polyamine, in particular EO„-b-MAA _m /polyamine in which n and m have the values designated above, and in particular having the formulas EOi ₇₇ -b-MAA ₂₃ /PEI or EOi ₈ 2-b-MAA ₃₅ /PEI.

7. Process for preparing a nanostructured material containing free pores according to one of claims 1 to 6, comprising a step of drying the mixture of organic polymer and micelles,

said drying step being carried out by evaporation of the organic solvent to obtain a dry film containing pores occupied by micelles, with a thickness of 1 μιη to 500 μιη, in particular of 10 μιη to 200 μιη, in particular of 50 μιη to 150 μιη.

8. Process for preparing a nanostructured material containing free pores according to claim 7, comprising a step of disassembling the micelles from the dry film, said disassembly of the micelles being carried out in particular by immersing the above dry film in an aqueous solution of pH > 8,

9. Process for preparing a nanostructured material containing free pores according to claim 8, comprising a step of drying, in particular under vacuum, a nanostructured material in the form of a film containing free pores after disassembly of the micelles,

to eliminate water molecules and obtain a nanostructured material containing free pores in the form of a dry film. 10. Process for preparing a nanostructured material containing micelles, comprising the following steps:

■ a step of mixing an organic polymer, in particular biodegradable, in a solvent, and micelles to obtain a stable mixture of organic polymer and micelles,

■ a step of drying the stable mixture of organic polymer, in particular biodegradable, and micelles, to eliminate the solvent and obtain a nanostructured material containing micelles in the form of a dry film containing pores occupied by micelles, said materials being obtained in the form dry film containing micelles with a thickness of 1 μιη to 500 μιη, in particular of 10 μιη to 200 μιη, in particular of 50 μιη to 150 μιη, ■ said organic polymer being in particular

° a polyester homopolymer, in particular a poly-L-lactide, a poly-D-lactide or a poly-D,L-lactide,

° or a block copolymer consisting of a block of polylactide in racemic form (poly-D,L-lactide, PDLLA) or in optically active dextrorotatory form (poly-D-lactide, PDLA) or levorotatory form (poly-L-lactide , PLLA), and a block chosen from polyethylene oxide PEO or polyglycolide,

said block copolymer having in particular the formula PEO-PDLLA, PEO-PDLA or PEO-PLLA,

° or a random copolymer, in particular biodegradable, consisting of polylactide in racemic form (poly-D,L-lactide, PDLLA) or in optically active dextrorotatory form (poly-D-lactide, PDLA) or levorotatory form (poly-L-lactide, PLLA), and polyglycolide,

said random copolymer being in particular poly-DL-lactide-polyglycolide,

■ said micelles being obtained by mixing two structuring parts in an aqueous solvent, the two structuring parts being a double-hydrophilic block copolymer containing a polyelectrolyte sequence and a polyelectrolyte of opposite charge,

said aqueous solvent having a pH of 5.0 to 7.0, in particular of 5.8 to 6.2, and in particular equal to 5.5 or 6.0,

° a structuring part being a double hydrophilic block copolymer (CBDH) consisting of polyethylene oxide (PEO) and poly(methacrylic acid) PMAA and being represented by the formula EO„-b-MAA _m ,

in which

• n designates the number of ethylene oxide units, n varying in particular from 50 to

800,

• m designates the number of methacrylic acid units, m varying in particular from

10 to 100,

· the n/m ratio being in particular between 3 and 10,

° the other structuring part being an organic polyamine chosen from polyethyleneimine PEI, chitosan, polyvinylpyridine or polyallylamine, in particular PEI or chitosan,

said polyamine possibly possessing therapeutic properties, and being in particular chitosan, lidocaine, tetracaine, procaine, cimetidine, gentamicin, kanamycin, doxorubicin, gencitabine, or a catecholamine, said micelles having the formula CBDH/polyamine, in particular EO„-b- MAA _m /polyamine in which n and m have the values designated above, and in particular having the formulas EOi ₇₇ -b-MAA ₂₃ /PEI or EOi ₈₂ -b-MAA ₃ 5/PEI,

the polymer/micelles ratio being in particular between 1 and 100, and in particular between 1 and 9,

to obtain a stable mixture of polymer and micelles in an organic solvent, the organic solvent used being chosen in particular from chloroform, chlorobenzene, dimethyl sulfoxide or toluene.

11. Process for preparing a nanostructured material containing micelles according to claim 10, comprising a step of drying a stable mixture of organic polymer, in particular biodegradable, and micelles,

and with a thickness of 1 μιη to 500 μιη, in particular of 10 μιη to 200 μιη, in particular of 50 μιη to 150 μιη.

12. Process for preparing a nanostructured material containing micelles according to one of claims 10 or 11, in which the micelles consist of a CBDH, in particular containing a polyelectrolyte sequence, in particular a polyanionic sequence, and a active principle containing an ionized group, in particular cationic, in particular an organic polyamine having therapeutic properties,

said polyamine constituting the active principle being chosen from chitosan, lidocaine, tetracaine, procaine, cimetidine, gentamicin, kanamycin, doxorubicin, gencitabine or a catecholamine.

13. Nanostructured material in the form of a dry film containing free pores occupied by micelles as obtained by implementing the method according to one of claims 10 to

12.

14. Nanostructured material containing free pores in the form of a dry film as obtained by implementing the method according to one of claims 1 to 9.