WO2010058132A1

WO2010058132A1 - Materials in the form of interpenetrating polymer networks combining a fibrin gel and a glycol polyethylene network

Info

Publication number: WO2010058132A1
Application number: PCT/FR2009/052224
Authority: WO
Inventors: Véronique LARRETA-GARDE; Odile Fichet; Amivi Edéfia AKPALO
Original assignee: Universite De Cergy-Pontoise
Priority date: 2008-11-20
Filing date: 2009-11-19
Publication date: 2010-05-27
Also published as: FR2938544A1; EP2358774A1; EP2358774B1; FR2938544B1; PT2358774E; DK2358774T3; ES2544763T3

Abstract

The present invention relates to a method for preparing a material in the form of an interpenetrating polymer network (IPN) combining a physical fibrin gel and a polyethylene glycol (PEG) network. The invention also relates to materials that can be obtained by said method as well as to the use thereof as dressings for wounds, surgical dressings, as a device for delivering therapeutic agents, as a covering for medical devices such as stents, heart valves, catheters, prosthetic vascular filters, etc. Said materials can also be used for eukaryotic cell cultures or even as a medium for active molecules such as antibiotics or enzymes.

Description

MATERIALS IN THE FORM OF INTERPENETRA NETWORKS

OF POLYMERS ASSOCIATING A FIBRIN GEL AND

A POLYETHYLENE GLYCOL NETWORK

DESCRIPTION

Technical area

The present invention relates to a method for preparing a material in the form of an interpenetrating network of polymers (RIP) combining a physical fibrin gel and a polyethylene glycol (PEG) network.

It also relates to the materials obtainable by this method as well as their use as dressing for wounds, surgical dressing, as a device for delivering therapeutic agents, as a coating for medical devices such as stents, valves for the heart, catheters, vascular prosthetic filters etc. Said materials can also be used for eukaryotic cell cultures, or as support for active molecules such as healing agents, growth factors, antibiotics, bactericidal agents, bacteriostatic agents or enzymes.

In the description below, references in square brackets [] refer to the list of references at the end of the text.

STATE OF THE ART Obtaining a material in the form of a gel remains a real need, particularly in the medical field. The gels possess the property of being elastic solids very rich in solvent. In particular, for biogels or gels obtained from molecules of biological origin, the solvent is water. Their volume and shape may vary reversibly depending on environmental stimuli such as pH, temperature, solvent composition, ionic strength, fields electric and magnetic. These biogels are hydrophilic networks that retain large amounts of water without dissolving and thus have similar physical characteristics to soft biological tissues. The importance of hydrogels (crosslinked polymers or biogels) in the field of biomaterials is justified. by the soft and elastomeric nature of hydrogels which minimizes mechanical irritation and friction with the tissues thus the inflammatory processes. Hydrogels have high permeability to oxygen, nutrients, and other water-soluble metabolites that promote the growth of thicker tissue structures [2]. Hydrogels are attractive for drug delivery because of their good compatibility with hydrophilic and macromolecular compounds (proteins, polysaccharides, oligonucleotides), their biocompatibility and the ease of regulating drug diffusion by controlling their swelling, crosslinking and their degradation. These materials are useful for localized diffusion since they can be formed in situ and thus adhere and conform to the target tissues. This use for local delivery of active ingredients seems to act in synergy with the barrier effect of hydrogels [3]. Hydrogels have been studied as potential materials for the regeneration of bone, tendon, nerves and essentially cartilage.

However, the gels formed from biological molecules are soft materials, also their main disadvantages are to have a low mechanical strength, to be very deformable and difficult to handle.

There is therefore still a real need to be able to prepare a material which can be handled, which has the properties of a gel of stable dimensions and improved resistance to degradation, for example by the action of heat or enzymes, etc. In the context of the present invention, a "network" is a combination of polymers. This association between the polymers can be ensured by strong or weak interactions (covalent bonds, hydrogen bonds, Van der Waals, etc.). In the case of covalent bonds, the combination is a crosslinking and the polymer is said to be "crosslinked".

The definition of a gel includes three criteria [4]:

- The gels are composed of at least two components: one very strongly majority is the solvent and the other, minority, is called solid.

- Both components are continuous throughout the environment. The so-called solid phase constitutes a network that traps the so-called liquid phase corresponding to the solvent, and prevents it from flowing.

- The whole medium behaves like a soft solid easy to deform.

Gels are classifiable according to the type of links that form the network. Thus two major gelling mechanisms are distinguished which lead to "physical" gels or "chemical" gels.

A physical gel is a supramolecular assembly consisting of molecules linked together by low energy bonds (Van der Waals, hydrogen bonds, polar bonds, etc.). The stability of this assembly is associated with a precise range of physico-chemical conditions (pH, concentration in molecules, temperature, quality of the solvent, ionic strength, etc.). Outside this range, the mixture is liquid. The sol / gel transition is therefore reversible for physical gels. Thus, a modification of the environmental parameters can lead to the destruction of the building and induce a freeze-soil transition. It is well known from the prior art of the compositions to obtain "physical" gels. Biogels are obtained essentially from macromolecules or polymers of natural origin: proteins or polysaccharides. Examples of proteins capable of gelling are gelatin, fibrin, collagen, actin, myoglobin, whey proteins, in particular caseins and lactoglobulin, proteins of soybeans, wheat and in particular gliadin and glutenin, egg white and in particular ovalbumin. Examples of polysaccharides that may gel carrageenans, alginates, pectins, chitosan, cellulose, chitin, glycogen or starch.

Also known in the state of the art gels called "chemical". A chemical gel corresponds to a supramolecular assembly whose molecules are associated by high energy (covalent) bonds. The stability of this assembly is very large. These chemical gels have improved stability, the only way to perform a gel / solution transition is to destroy the covalent bonds of the network. This is why the ground / gel transition of chemical gels is called irreversible. A family of chemical gels corresponds to enzymatically catalyzed gels. This mode of gelation is mainly observed in large biological processes. Blood coagulation, scarring, skin formation, extracellular matrix assembly are biological processes where the passage of soluble proteins in the gel state is essential. In vivo, a limited number of enzymes, for example lysyloxidases, transglutaminases, catalyze these reactions. In vitro, the most commonly used is transglutaminase which creates covalent bridges between the side chains of lysine and glutamine residues of proteins [5].

The structural and biochemical properties of fibrin make it a promising candidate in tissue engineering and regenerative medicine.

The modification and functionalization of fibrin gel has been used for the controlled release of genes and growth factors. In addition, fibrin naturally has cell binding sites and has therefore also been studied for adhesion, spreading, migration and cell proliferation. Because of proven effects on several types Fibrin is of particular interest as a support for vascular tissue engineering [6].

Fibrin is obtained from fibrinogen, a 340 kDa glycoprotein of hepatic origin. Fibrinogen is an anionic polyelectrolyte under physiological conditions and therefore can not associate or aggregate. The hydrolysis of fibrinogen by thrombin, a serum protease, leads to the release of small peptide fragments and the production of fibrin. The intermolecular interactions then become possible, the molecules self-associate via low energy bonds, thus creating a network of physical fibrin gel.

In the process of coagulation or healing, the stabilization of the physical gel is achieved by a serum transglutaminase (factor 13) creating covalent isopeptide bonds between the fibrin chains leading to the formation of an insoluble fibrin chemical network. However, chemical fibrin gels are soft materials, so their main disadvantages are to have a low mechanical strength, to be very deformable and difficult to handle.

As indicated above, there is a need for a material in the form of gel called "physical", which is stable and maintains its structural integrity and therefore its properties for the duration of the use made of it, but whose elements of biological origin are reversible (or labile) that is to say that can be destructured and degraded when desired. To date, most known methods allow the preparation of material in the form of gel called "chemical". One possibility for obtaining a self-supported gel having these properties is to introduce this gel into an interpenetrating or semi-interpenetrating network architecture of polymers (RIP or semi-RIP).

There is therefore a real need for a method that overcomes the shortcomings, deficiencies, limitations, disadvantages and obstacles of the state of the art, in particular of a process using a material in the form of interpenetrating network of polymers (RIP) combining a physical fibrin gel and a polyethylene glycol network.

This process allows the preparation of said interpenetrating network of polymers (RIP) in a step ("one pot" in English) under operating conditions respecting the nature of each polymer thus avoiding their degradation.

Moreover, this method makes it possible to control the formation of said network in situ, thus being able to provide a "made-to-measure" material adapted to the application for which it is intended.

Description of the invention

The present invention is specifically intended to meet this need by providing a method for preparing a material in the form of an interpenetrating network of polymers (RIP) combining a physical fibrin gel and a polyethylene glycol (PEG) network, comprising the following steps: i) prepare a mixture by introducing into buffer

a solution of fibrinogen,

monomers of formula (I): Xr (CH ₂ -CH ₂ -O) _n -X ₂ in which X ₁ and X ₂ are identical or different chemical groups chosen from the group comprising vinyl, acrylate, methacrylate or allyl and n is a number between 1 and 140, for example from 1 to 50, preferably from 2 to 20,

optionally monomers of formula (II) chosen from polyethylene glycol derivatives bearing a group chosen from the group comprising -CH ₃ , -NH ₂ , -OH, -CH ₂ CH ₃ , vinyl, acrylate, methacrylate or allyl; acrylate, methacrylate and styrene derivatives selected from the group consisting of hydroxyethylacrylate, hydroxyethylacrylate, vinyl acetate, N-vinylpyrrolidone, N-vinylpyridine, acrylonitrile, acrylic acid, sodium styrene sulfonate, (vinylbenzyl) trimethylammonium bromide, (vinylbenzyl) trimethylammonium chloride, trimethylvinyloxycarbonylmethylammonium chloride, trimethylvinyloxycarbonylmethylammonium bromide, triethyl-2-vinyloxycarbonylethylammonium bromide, allyloxycarbonylmethyltrimethylammonium bromide, poly (ethylene glycol) methacrylate, poly (ethylene glycol) acrylate,

a polymerization initiator; ii) preparing a reaction mixture by adding a thrombin solution to the mixture prepared in i); iii) incubating the reaction mixture obtained in ii) at a temperature between 20 and 40 ⁰ C; iv) polymerization and crosslinking of the monomers of formula (I) with optionally the monomers of formula (II).

The process of the invention makes it possible to prepare a material in the form of an interpenetrating network of polymers (RIP) which is homogeneous, manipulable, stable over time and of stable dimensions.

The process according to the invention, in particular through the temperature of the incubation and the polymerization, makes it possible to control the gelation and the polymerization / crosslinking of the monomers. Gelation and crosslinking may occur simultaneously or sequentially. Furthermore, when the polymerization is a photopolymerization, the material obtained at the synthesis output (at the end of step (iv)) is advantageously a sterile material.

In the context of the present invention, the terms polyethylene glycol (PEG), polyethylene oxide or polyoxyethylene (POE) are indifferently used to designate the polyethylene glycol network obtained by crosslinking a monomer mixture of formula (I) or by polymerization / crosslinking of a monomer mixture of formulas (I) and (II).

The monomers of formulas (I) can be described as difunctional (carrying two reactive functions according to the proposed crosslinking mechanism). The monomers of formula (II) are monofunctional (a single reactive function according to the proposed polymerization mechanism).

In the context of the present invention, the term "buffer" means an aqueous solution whose water content is at least 30% by weight up to 99% by total weight of the solvent and which maintains approximately the same pH despite the addition of small amounts of an acid, a base or a dilution. The water content of the buffer is in particular 70 to 98% by total weight of the solvent.

The buffer may further comprise other solvents selected from the group consisting of methanol, ethanol, pyridine, acetone, acetic acid, DMSO, benzene, dichloromethane.

By way of example of a buffer, mention may be made of phosphate, Hepes, Tris, sodium barbital and Tris-maleate buffers.

Throughout the duration of the process, the pH of the aqueous medium is maintained between 6 and 8 by means of a buffer solution, for example a solution of trihydroxyaminomethane (Tris).

The fibrinogen can be, for example, human fibrinogen, pork or beef.

In the process of the invention, the monomers of formulas (I) may be used alone or as a mixture with monomers (II) as defined above.

In the case of the monomers of formula (I), n can be defined as the average number of oxyethylene units (-CH ₂ CH ₂ O-) contained in said monomers. Preferably, the monomers of formula (I) are those in which

Xi and X ₂ , are identical or different chemical functions, chosen in the group comprising methacrylates or acrylates and n is a number between 2 and 20.

The monomers of formula (I) may, for example, be used alone. These monomers are precursors of the polyethylene glycol (PEG) network.

The (PEG) network may contain between 1 and 100% by weight of monomers of formula (I) and between 99 and 0% by weight of monomers of formula (II). The network (PEG) may for example contain between 10 and 100% by weight of monomers of formula (I) and between 90 and 0% by weight of monomers of formula (II).

In a particular embodiment of the invention, when it is desired to have a material having bactericidal or bacteriostatic properties, the monomer of formula (II) may advantageously carry an ammonium group. Any initiator, free radical generator known to those skilled in the art, for the polymerization may be suitable.

For example, the initiator may be a thermal initiator selected from the group consisting of peroxides, diazo compounds or persulfates, for example potassium persulfate, ammonium persulfate and hydrogen peroxide.

For example, the initiator may be a photopolymerization initiator. It can be chosen from the group comprising Irgacures (for example 2959, 184, 651, 819) [6], 2-hydroxy-4- (2-hydroxyethoxy) -2-methylpropiophenone, benzophenone, 2, A- dimethylbenzophenone, benzoin, ionic benzophenones selected from the group consisting of 4-trimethylmethylammonium benzophenone chloride, 4-sulfomethylbenzyl sodium salt, aromatic ketones and aldehydes including benzaldehyde, acetophenone, biacetyl, parachlorobenzophenone, ferrulic acid, and all compounds producing UV / visible radiations. Irgacure 2959 is preferably chosen. The concentrations of the various compounds in the mixture (stage i) can be for example:

for fibrinogen of 1 to 50 mg / ml, preferably between 5 and 25 mg / ml, for the monomers of formula (I) and optionally (II) of 1 to 400 mg / ml, preferably 100 mg / ml from 0.1 to 1 mg / ml, preferably 0.42 mg / ml,

The thrombin used in step (ii) may be derived for example from human plasma, pork or beef.

The concentration of thrombin in the mixture of step (ii) may be, for example, from 0.1 to 2.5 U / mL, preferably from 0.2 to 1 U / mL.

In step iii), the incubation temperature may be, for example, between 30 and 40 ^° C., preferably 37 ° C.

It is during this step that the physical fibrin gel is formed from fibrinogen by enzymatic hydrolysis. This enzymatic hydrolysis is catalyzed by thrombin. Unexpectedly, the formation of the gel, the activity of the enzyme and the properties of the gel are not impaired by the presence of the monomers of formula (I) and optionally monomers of formula (II), and their polymerization.

When the initiator is a thermal initiator, the polymerization of the monomers of formulas (I) and / or (II) can take place at the same time as step (iii) or after step (iii). When the polymerization takes place after step (iv), it may be carried out for example at a temperature of between 25 and 60 ^° C. Preferably, the temperature is chosen so that the fibrin gel is not denatured. .

The thermal polymerization can be carried out between 0.5 and 10 hours, preferably between 1 and 3 hours. In step iv), when the initiator is a photopolymerization initiator, for example those mentioned above, the polymerization can be carried out under UV / visible radiation at a wavelength between 190 and 800 nm, preferably under UV radiation with a wavelength of between 300 and 380 nm.

The photopolymerization in step iv) can be carried out for 1 to 10 hours, preferably 1 hour and 30 minutes to 3 hours.

The different solutions are prepared in Tris buffer or

HEPES, preferably Tris-HCl, at a concentration of 25 to 100 mM, preferably 50 mM, at a pH of 7.2 to 7.5, preferably 7.4

CaCl ₂ from 10 to 50 mM, preferably 20 mM and NaCl from 50 to 250 mM, preferably 150 mM.

The final concentrations of the various compounds in the mixture (step ii) can be for example:

for fibrinogen of 1 to 50 mg / ml, preferably between 5 and 25 mg / ml, for the monomers of formula (I) and optionally (II) of 1 to 400 mg / ml, preferably 100 mg / ml ,

for the initiator from 0.1 to 1 mg / ml, preferably 0.42 mg / ml, and

for thrombin from 0.1 to 2.5 U / mL, preferably from 0.2 to 1 U / mL.

After step (iv), the method of the invention may further comprise a step of enzymatically crosslinking the physical fibrin gel. In this additional step, for example transglutaminase or lysyloxidase can be used as the enzyme.

Transglutaminase is an enzyme that covalently bonds (or connects) certain amino acids to one another (essentially glutamic acid and lysine) and contributes to the crosslinking of proteins during gelation processes. This can lead to a more biodestruction-resistant system with improved mechanical properties.

According to this embodiment, the enzyme, for example transglutaminase, may be used. The RIP sample obtained after step (iv) is then immersed immediately or later in a transglutaminase solution at a concentration ranging from 0.1 to 10 U / mL, preferably between 0.5 and 3 U / mL for varying times of 5 minutes to 6 hours, preferably 30 minutes to 3 hours.

The process of the invention can be implemented in any container, beaker, crystallizer, pill, etc. preferably can be closed. The container can also be made to measure with, for example, two sheets of glass or any material transparent to UV and impervious to water. These plates may be separated by a Teflon ^D or polyethylene gasket of different thicknesses from 100 Dm to 3 mm, preferably 1 mm.

The invention also relates to the material in the form of an interpenetrating network of polymers (RIP) combining a physical fibrin gel and a polyethylene glycol (PEG) network that can be obtained by the method according to the invention. The material obtainable by the process according to the invention has the advantage of being in the form of a gel, homogeneous and manipulable while exhibiting dimensional stability over time and capable of withstanding degradation, for example by action of heat. In addition, the resistance to degradation of said material under the action of enzymes is significantly improved.

As indicated above, the material of the invention is in the form of an interpenetrating network of polymers (RIP) combining a physical fibrin gel and a polyethylene glycol (PEG) network between which there is no chemical bond, it is ie no covalent bond. In this interpenetrating network of polymers (RIP), each polymer forms a chemical or physical network having a three-dimensional or 3D existence. For example, the PEG network may be synthesized by crosslinking poly (ethylene glycol) di (meth) acrylate (X ₁ = X ₂ = (meth) acrylate where n can vary from 2 to 140). The PEG network swells in water or buffer. According to an advantageous embodiment of the invention, in the material, the interpenetrating network of polymers (RIP) associates a gel fibrin physics and a polyethylene glycol network made with poly (ethylene glycol) dimethacrylate (Xi = X ₂ = methacrylate and n = 8 to 9 or n = 13 to 14).

According to another advantageous embodiment of the invention, it is possible to synthesize the polyethylene glycol network with poly (ethylene glycol) diacrylate (Xi = X ₂ = acrylate and n = 8 to 9 or n = 13 to 14) .

In the material, the polyethylene glycol (PEG) network may contain between 1 and 100% of monomers of formula (I) and between 99 and 0% by weight of monomers of formula (II) as defined above.

More particularly, the monomers of formula (I) are those in which X ₁ and X ₂ , which are identical or different, are used alone and are chosen from the group comprising methacrylates or acrylates and n is a number between 2 and 20. The material of the invention may consist of: a) from 1 to 50% by dry weight of fibrin; b) from 50 to 99% by dry weight of a polyethylene glycol (PEG) network.

The material according to the invention has improved mechanical properties compared to those of a gel consisting solely of fibrin.

It also has (quasi-) reversible properties of dehydration and hydration. This is not the case, for example, for the fibrin gel alone. Indeed, after a dehydration cycle, the fibrin gel can not rehydrate more than 25% of the initial value. The material according to the invention can be rehydrated from 25 to 100%, preferably from 45 to 95%.

Moreover, the material according to the invention has a significantly improved resistance to degradation, for example: by proteases, by temperature, etc. Fibrin is stabilized in this material. Indeed, when a fibrin gel is immersed in buffer

Tris (50 mM pH 7.4) for 48 h, 25% of the protein is extracted. In the RIP this proportion is between 20 and 0.1% usually between 1 and 0.1%. The polyethylene glycol (PEG) network appears to "protect" fibrin from degradation.

The material according to the invention has a glass transition temperature (Tg) of between -100 and +100 ^° C., preferably between -50 and

+20 ^° C. The glass transition temperature (Tg) of a polymer material is defined as the temperature below which the polymer chains have a low relative mobility. Below Tg the polymer is in a glassy state, above Tg, the polymer is in a rubbery state. The glass transition temperature of a material can be measured, for example, by differential scanning calorimetry DSC.

The material of the invention may also have a storage modulus (measured in shear mode, G ') of between 100 Pa and

10 MPa, for example between 1000 Pa and 6 MPa, in the hydrated state at 37 ° C. and a storage modulus (measured in voltage mode E ') of between 0.01 and 3000 MPa, for example between 1 and 3000 MPa, in the dry state.

The modulus X of a material subjected to sinusoidal stress is written in the complex form:

X * = X '+ iX "

According to the stressing mode used, voltage or shear, the modules X are denoted respectively E or G, X 'are denoted E' or G 'and X "are denoted E" or G ", i is a complex number such that (i Q = -1 [7] In voltage mode, the real part of the module E ', elastic or conservation modulus, measures the capacity of the material to store energy whereas the imaginary part E ", viscous modulus or loss, represents the ability of the material to dissipate energy. Depending on the state in which the material is in function of the temperature, a phase shift D appears between the stress exerted on the material and its deformation. This phase shift D is zero when the material is purely elastic, and 90 ° when it is perfectly viscous. Thus, the tangent of the phase angle (tan D) reflects the damping capacity of the material.

To characterize a material, the spectra of ATMD (dynamic thermomechanical analysis) representing E 'or tan ^ as a function of temperature are recorded.

In shear mode, the modulus of conservation or elasticity in shear (G ') makes it possible to estimate the elasticity of the gel. The loss modulus or viscous modulus (G ") characterizes the liquid phase of the gel When G"> G ', the sample is considered as liquid and when G'> G ", the sample is gelled. gelation are characterized by a gel time resulting in an equality of modules G 'and G ".

The material that can be obtained according to the process of the invention is advantageously biocompatible. When it is desired to obtain a sterile final material at the synthesis output (at the end of the implementation of the process of the invention), the material is advantageously obtained by photopolymerization.

Another subject of the invention relates to the use of a material according to the invention or capable of being obtained according to the process of the invention, such as:

- dressing for wounds,

- surgical dressing,

a device for delivering therapeutic agents,

coating for medical devices selected from the group consisting of stents, heart valves, catheters, vascular prosthetic filters,

support of active molecules selected from the group comprising growth factors, antibiotics, bactericidal, bacteriostatic or enzymes, or for eukaryotic cell cultures. Other advantages may still appear to those skilled in the art on reading the examples below, illustrated by the appended figures, given for illustrative purposes.

Brief description of the figures

FIG. 1 represents the photos just after step iv) of the RIPs obtained from monomer mixtures (at the final concentration 100mg / nnL) of formula Xr (CH ₂ -CH ₂ -O) _n -X ₂ in which X ₁ and X ₂ are methacrylate groups and: - where n = 8 to 9 and the fibrinogen is at the final concentration 5 mg / mL

(RIP550-5)

where n where n = 13 to 14 and the fibrinogen is at the final concentration 5 mg / mL (RIP750-5)

where n = 8 to 9 and the fibrinogen is at the final concentration 25 mg / mL (RIP550-25)

where n = 13 to 14 and the fibrinogen is at the final concentration 25 mg / mL (RIP750-25).

FIG. 2 shows the absorption spectra measured with a Fourier transform infrared spectrometer equipped with an attenuated total reflection module (ATR) of RIP 550-5 (a) and RIP 750/5 (b). . In abscissa "NO" represents "Number of Wave" and in ordinate "TR" represents "Transmittance".

Figure 3 shows the dehydration isotherm of the different RIPs measured with a Thermogravimetric Analyzer (ATG) under argon flow at 37 ° C. (D: RIP 550-5; D: RIP 550-25; D: RIP 750-5; D:

RIP 750-25). On the abscissa "t" represents "time" and on the y-axis "PM" represents "Mass Loss".

Figure 4 shows the hydration-dehydration cycles of the RIPs 550-5 (D), RIP 550-25 (D), RIP 750-5 (D) and RIP 750-25 (D). On the abscissa "CG-S" represents "Swelling-Drying Cycles" and on the y-axis "VM" represents "Mass Variation". FIG. 5 represents the dynamic thermomechanical analysis (ATMD) in voltage mode as a function of the temperature of the simple network POE 550 (synthesized under the same conditions without fibrinogen in the mixture (i), RIP 550-5 and RIP 550 -25 dried a- Conservation Modules (MC), from bottom to top: RIP 550-5, simple POE 550 network, RIP 550-25 On the ordinate "T" represents "Temperature" b- Tan Delta (TN) from top to bottom: POE 550, RIP 550-5, RIP 550- 25. On the ordinate "T" represents "Temperature" D Figure 6 shows the voltage conservation modules

(TM) as a function of temperature (T) of RIP 550-25 (top) and RIP 750-25 (bottom) - In the figure inserted: Tan Delta - from top to bottom: RIP 750-25, RIP 550-25.

EXAMPLES

L Characterization of materials

THE. Characterization of mechanical properties by dynamic thermomechanical analysis (ATMD)

The thermomechanical characterizations in voltage mode of the different materials (previously dried 24h at 37 ° C in an oven) are measured by ATMD on a Q800 instrument (TA Instruments), with stress in voltage mode. The imposed deformation is 0.05%

(low reversible deformation), pre-tension force of 120%. The samples are biased at a constant frequency of 1 Hz with a temperature ramp of T = -80 ^° C. at 100 ^° C. at a heating rate of 5 ° C./min. The rectangular samples have variable length and width sizes but an average thickness of 0.1 mm. LB. Thermal analyzes

The differential scanning calorimetry (DSC) measurements were performed with a DSC Q100 instrument (TA Instruments) and the analysis software is Universal Analysis. The dried sample weighing less than 10 mg is placed in an aluminum capsule which is sealed and placed in the oven. The sample is cooled to 5 ° C / min until

-80 ⁰ C and a isotherm of one minute performed. Then a heater to

5 ° C / min is carried out up to 200 ^° C. (first sweep), followed again by an isotherm of one minute. A second pass is made on the same sample. The thermograms recorded at the second pass are analyzed to determine the glass transition temperatures.

LC. Water behavior of LC.1 materials. Dehydration Isotherms The thermogravimetric analyzes (ATG) are performed on a Q50 model of the company TA Instruments and the analysis software is Universal Analysis. The mass change of a sample is measured as a function of temperature and time under an argon atmosphere.

A mass m of hydrated sample is deposited in the nacelle, then the oven is closed. During the isothermal recordings, the temperature is equilibrated at 37 ° C., then maintained at this temperature for 100 minutes, under an argon atmosphere (60 ml.min ^-1 ).

IC2. Moisture-dehydration cycles The samples just after step (iv) are immersed for 2 hours in 25 ml of Tris-HCl buffer pH 7.4 containing 0.02% of sodium azide (NaN ₃ ) in order to achieve the maximum swelling rate that will correspond to a mass mi at the T1 point. Then the samples undergo 3 alternating cycles of dehydration - hydration. The dehydration experiments are carried out for 24 hours at 37 ° C in an oven (mass m ₂ , m ₄ and m ₆ corresponding to the points T2, T4 and T6) and those of rehydration are made in Tris buffer for 2 hours at the same temperature (mass m ₃ and m ₅ corresponding to the points T3 and T5). The last step leading to the mass m ₇ (point T7) is a rehydration in the Tris-HCl buffer of 24h at 37 ° C.

From the masses obtained at each step Ti, the hydration - dehydration curves are plotted by representing the variation

Tn ₁ U m ₁

Um = relative mass ^ι according to the number of the step carried out.

LD. Spectroscopic analyzes Absorbance measurements are performed on a spectrophotometer

UVIKON XS double beam from the company SECOMAM.

The infrared spectra are recorded on a Tensor 27 IR spectrophotometer equipped with an ATR reflection module (45 ° angle diamond cone).

THE. ELISA tests

The ELISA assays, for quantifying the amount of protein extracted from the materials, are performed as follows: extractions are made by immersing a 1 mL sample of fibrin gel or RIP in 4 mL of cover buffer overnight at 37 ° vs. The solution containing the extractables is diluted 100-fold (with the exception of post-polymerizations with transglutaminase) before the recovery of the wells. For each result the experiment is performed in n = 3 and at least 3 times. The buffers used are based on TBS buffer (Tris saline buffer) composed of 10 mM Tris 1 and 1.65 M NaCl at pH 7.4. The binding buffer is composed of 10 mM Tris, 150 mM NaCl and 0.02% NaN _3. The saturation buffer is composed of TBS containing 5% milk powder and 0.1% Tween-20. The rinse and dilution buffer is composed of TBS containing 0.05% Tween-20 and 0.02% NaN ₃ . Finally, pNPP is dissolved in 1 M diethanolamine containing 0.5 mM MgCl ₂ at pH 9.8. The first step of the ELISA test consists in fixing the antigen by incubating 100 μl of the extractable in each of the wells for 3 hours at 37 ° C. After three rinses with 200 μl of rinsing buffer, 200 μl of saturation buffer are added to the wells, the whole is incubated overnight at 4 ° C. After four rinses with 200 μl of rinse buffer, 100 μl of the primary antibody diluted to 1/200 (human anti-fibrinogen produced in the rabbit) are introduced into the wells and the whole is incubated for 3 hours at 37 ° C. vs. After rinsing, 100 μl of the secondary antibody diluted to 1/30 000 th (anti-immunoglobulin G from rabbit produced in the goat and coupled to alkaline phosphatase) are added, the whole is incubated for 3 hours at 37 ° C. After rinsing, 200 μl of 1 mg / nnL pNPP (the chromogenic substrate) are added. The development of the coloration is done in the dark for 15 minutes and is stopped by 50 μl of 3M NaOH. The measurement of the colorimetric assay is carried out at 405 nm, the intensity of the signal is directly proportional to the fibrinogen concentration.

LF. Solid-liquid extraction The extraction with dichloromethane in a soxhlet makes it possible to solubilize all the compounds soluble in this solvent (thus non-crosslinked) and to quantify their proportion in a solid sample.

The amount of soluble material contained in the RIPs corresponds to the proportion of uncrosslinked PEGDM monomer. The materials are extracted with soxhlet with CH ₂ Cl ₂ at reflux for 48 hours. The extractible rates are calculated according to the ratio of masses:

(m Y \ m _f ) Mat. Ext. % = ' ^f xl 00 m>, where m, and nri _f are the masses of the materials before and after extraction, respectively. The extractions were carried out on samples containing 100 mg of POE network, that is to say that m, = 100 mg. IL Characteristics of the compounds used

Sodium chloride (NaCl - CAS 27 800.291 Research organics), calcium dichloride (CaCl ₂ , 2H ₂ O - CAS 22 317.297 - Research organics), hydrochloric acid (37% HCI - CAS 20 252.290 - Prolabo), urea (CAS 28876.367 - Prolabo), sodium dodecyl sulphate (SDS -

CAS 16103.01 - Biorad), oMercaptoethanol (CAS 16107.10 - Biorad),

Diethanolamine (C ₄ HnNO ₂ - CAS 111 -42-2), Tween-20

Polyoxyethylene-Sorbitan Monolaurate (CAS 9005-64-5) and sodium azide (NaN ₃ - CAS 26628-22-8) are from Sigma. The various poly (ethylene oxide) dimethacrylate (PEGDM - CAS 25852-47-

5 - Aldrich) of molecular weights 330 (PEGDM330), 550 (PEGDM550) and

750 (PEGDM750) g.mol ^"1 , monofunctional poly (ethylene glycol) methacrylate (530 gmol ^{" 1} - ALDRICH), 2-hydroxy-4- (2-hydroxyethoxy) -2-methylpropiophenone (Irgacure 2959 - CIBA ), dichloromethane (Carlo-Erba), trihydroxyaminomethane (Tris, Prolabo or

Carlo Erba), are used without prior treatment.

P-Nitrophenyl Phosphate (pNPP) and iodoacetamide (1-6185 - CAS 144-48-9) come from SIGMA. This chromogenic substrate (EC 224-246-5) is in tablet and in the form of 5mg tablets.

Fibrinogen type IV (Fg, F-4753 - SIGMA) used is of bovine origin. The lyophilized protein is stored at -20 ^° C. and is kept at this same temperature after solubilization.

Lyophilized thrombin (EC3.421.5 - 34.8 unit per mg of solid - SIGMA) is stored at -20 ^° C. Its solution in Tris-HCl buffer can be stored for 2 to 3 days at + 4 ° C and -20 ^° C. for a longer duration.

Transglutaminase (Activa WM - Ajinomoto company distributed by UNIPEX), packaged in powder, has an activity of 100 units per gram of solid and is stored at -20 ° C. Thermolysin (SIGMA) has 33 units per mg of solid and is stored at -20 ° C. For the ELISA experiments, the human anti-human fibrinogen primary antibody produced in the rabbit comes from the company DAKO (CAS-A 0080). The rabbit anti-IgG (immunoglobulin G-A 3687-Sigma) secondary antibody is produced in the goat and coupled to alkaline phosphatase.

All solutions are prepared in 50 mM Tris-HCl buffer pH 7.4. Stock solutions at 2M CaCl ₂ , 1, 5M NaCl as well as 10mg / ml and 50mg / ml fibrinogen are prepared. The stock solutions of thrombin and transglutaminase are respectively prepared at 20 units / ml and 10 units / ml in tris-HCl buffer.

III. Examples of material development and their characterization Example 1: Synthesis of RIP POE550 / Fibrin (100/5) (RIP550-25)

Stock solutions of fibrinogen (10 mg / mL), thrombin (20 U / mL), NaCl (1.5 M), CaCl ₂ (2 M), and Irgacure 2959 (6 mg / mL) prepared in Tris-HCl buffer (50 mM, pH 7.4) and the pure PEGDM550 monomer are incubated separately for 15 minutes at 37 ° C in a water bath.

210 μl of Tris-HCl buffer, 10 μl of CaCl ₂ (2 M), 100 μl of NaCl (1.5 M), 500 μl of fibrinogen (10 mg / ml), 100 μl of monomer PEGDM 550 and 70 μl of Irgacure 2959 (6 mg / ml) is added in this order to a 1.5 ml ependorff tube. The gelation reaction of the fibrin is initiated by the addition of 10 μl of a thrombin solution in the buffer

Tris-HCl at a concentration of 20 units / mL. The reaction mixture of a total volume of 1 ml is pipetted homogenized and rapidly introduced between two glass slides separated by a Teflon seal (registered trademark) with an average thickness of 1 mm. These blades held by clamps are deposited on a support in a water bath maintained at 37 ° C. The radical polymerization is carried out during 1 h30 min away from daylight with a type VL-6 lamp with a power of 2D5W emitting UV radiation at D = 365 nm.

RIP 550-5 is a soft deformable material in the form of a rectangle of 6.5 cm x 2 cm and an average thickness of 1 mm (Figure 1).

RIP 550-5 contains a dichloromethane-soluble fraction of 36% by weight with respect to the mass of PEGDM550 monomer initially introduced. 10% of the protein mass can be extracted from this RIP. The infra-red spectrum of the RIP is reported (Figure 2). Its dehydration behaviors under argon flow at 37 ° C (measured by ATG), on the one hand, and the hydration-dehydration cycles, on the other hand, are reported, respectively (Figure 3 and Figure 4 ).

RIP 550-5 has a glass transition temperature (Tg) detected at -37 ° C by DSC measurement during the second scan. RIP 550-5 exhibits a shear mode conservation modulus of the order of 0.8 MPa in the hydrated state (measured in ATMD in shear mode). The thermomechanical properties of this RIP in the dried state are reported (Figure 5).

Example 2 Synthesis of RIP POE550 / Fibrin (100/25) (RIP 550-

25)

RIP 550-25 (FIG. 1) is synthesized according to the same protocol as that described in Example 1. Only the concentration of the stock solution of fibrinogen is set at 25 mg / ml, all the other concentrations remain identical. The volumes introduced are the same.

RIP POE550 / Fibrin (100/25) does not contain a dichloromethane-soluble fraction (0% dichloromethane extractable). The ELISA tests show that 1% by weight of introduced protein is extracted from the RIP. This RIP has a glass transition temperature (Tg) detected by DSC at -27 ° C. His dehydration behavior under argon flow at

37 ° C (measured by ATG), on the one hand, and hydration cycles- dehydration, on the other hand, are reported, respectively (Figure 3 and Figure 4).

RIP 550-25 has a shear modulus of the order of 0.3 MPa in the hydrated state (measured in ATMD in shear mode). The thermomechanical properties of this RIP in the dried state are reported (Figure 5 and Figure 6).

Example 3 Synthesis of RIP POE750 / Fibrin (100/5) (RIP 750-5)

RIP 750-5 is synthesized according to the same protocol as that described in Example 1. Only the monomer PEGDM 550 is replaced by the monomer PEGDM 750. The concentrations and volumes introduced are the same.

The RIP 750-5 is rigid and brittle: it can be cut with a pipette tip (Figure 1). It crackles during drying. It contains a dichloromethane-soluble fraction of 24% by weight relative to the mass of monomer PEGDM750 introduced. 25% of the protein mass can be extracted from this RIP. This RIP has a glass transition temperature (Tg) detected by DSC at -38 ° C. The infra-red spectrum of the RIP is reported (Figure 2). Its behavior under argon dewatering at 37 ° C (measured by ATG), on the one hand, and the hydration-dehydration cycles, on the other hand, are reported, respectively (Figure 3 and Figure 4). .

Example 4 Synthesis of RIP POE750 / Fibrin (100/25) (RIP 750-25)

RIP 750-25 (FIG. 1) is synthesized according to the same protocol as that described in Example 2. Only the monomer PEGDM 550 is replaced by the monomer PEGDM 750. The concentrations and volumes introduced are the same. RIP 750-25 contains 0% dichloromethane soluble fraction.

ELISA tests show that 1% by weight of protein introduced is extracted from the RIP. Its behavior under argon flow dehydration at 37 ° C (measured by ATG), on the one hand, and the hydration-dehydration cycles, on the other hand, are reported, respectively (Figure 3 and Figure 4). . RIP 750-25 has a shear modulus of the order of 1, 2

MPa in the hydrated state (measured in ATMD in shear mode). The thermomechanical properties of this RIP in the dried state are reported (Figure 6).

EXAMPLE 5 Enzymatic Postcrosslinking of the Fibrin Network in the RIP POE550 / Fibrin (100/5) (RIP 550-5)

The RIP 550-5 synthesized according to the protocol described in Example 1 is immersed for 3 hours in 10 ml of a transglutaminase solution at 0.5 units / ml in Tris-HCl buffer. ELISA tests show that less than 0.2% of the introduced protein mass can be extracted from this RIP.

Example 6: Enzymatic postcrosslinking of the fibrin network in the RIP POE550 / Fibrin (100/25) (RIP 550-25) The RIP 550-25 synthesized according to the protocol described in Example 2 is immersed for 3 hours in 10 minutes. mL of a transglutaminase solution at 0.5 units / mL in Tris-HCl buffer. ELISA tests show that 0.2% of the introduced protein mass can be extracted from this RIP. This material has a shear modulus of the order of 1 MPa in the hydrated state (measured in ATMD shear mode at 1 Hz).

Example 7 Enzymatic Postcrosslinking of the Fibrin Network in the RIP POE750 / Fibrin (100/5) (RIP 750-5)

The RIP 750-5 synthesized according to the protocol described in Example 3 is immersed for 3 hours in 10 ml of a transglutaminase solution at 0.5 units / ml in Tris-HCl buffer. ELISA tests show that 0.2% of the introduced protein mass can be extracted from this RIP.

Example 8 Enzymatic Postcrosslinking of the Fibrin Network in the RIP POE750 / Fibrin (100/25) (RIP 750-25)

The RIP 750-25 synthesized according to the protocol described in Example 4 is immersed for 3 hours in 10 ml of a 0.5 units / ml transglutaminase solution in Tris-HCl buffer. ELISA tests show that 0.2% of the introduced protein mass can be extracted from this RIP. This material has a shear modulus of the order of 3 MPa in the hydrated state (measured in ATMD shear mode at 1 Hz).

Example 9 Biocompatibility of RIPs

CHOK1 cells were cultured on RIPs 750-5 and RIP 750/25 described respectively in Example 3 and Example 4. For both materials:

• the cells adhere to the materials, especially on the RIP 750/25.

• DAPI nuclei labeling (4 ', 6-diamino-2-phenyl-indole) shows that the cells are alive after at least 5 days. • observation in fluorescence microcopy shows that cells divide after adhering to the material

• a fluorescent labeling of the actin network proves that the cells are spread over the materials.

• several cells could be observed during the mitosis phase. The cells multiply on the materials.

• cells tend to penetrate the material especially for the RIP 750/25

All these observations lead to the conclusion that RIP 750-5 and RIP 750/25 are biocompatible. Example 10: Capacity of Retention / Release of Molecules

The RIP 750/25 described in Example 4 was rinsed abundantly at the synthesis output, then immersed in a culture medium adapted to CHOK1, dried with paper towel, then seeded into eukaryotic cells. The cells adhere and exhibit greater cellular activity on this material than on the same "non-enriched" material described in Example 10.

Example 11 Molecular Retention / Release Capacity The RIP 750/25 described in Example 4 is rinsed with Tris buffer.

HCI synthesis output, dried with absorbent paper, then immersed in an antibiotic solution and dried with absorbent paper. Three different antibiotics: chloramphenicol, streptomycin and ampicillin were used. These materials are then deposited on a petri dish covered with bacteria. After 24 hours, in each case, an abiotic zone (without presence of life) is observed showing that the antibiotic has been released by the material. All controls where the material is alone are negative.

The RIP 750/25 thus has capacity for retention / release of molecules active ingredients.

Example 12 Comparison with other oligomers

The feasibility of other RIPs and their mechanical properties were verified by first varying the chain lengths of the PEGDM oligomer, ie the crosslinking density of the POE network synthesized in the RIP.

PEGDM 330 with a lower molar mass (330 g mol ^-1 ) than PEGDM 550 was chosen A synthetic monomer concentration of 100 mg / ml is retained PEGDM 330 is not sufficiently soluble in the water. water (solubility: 65 mg / mL), its 100 mg / mL solution in the Tris-HCl buffer is therefore not homogeneous and the synthesis of the RIP logically results in an inhomogeneous material.

POE networks synthesized from 10/90 and 90/10 mixtures by weight of PEGDM 550 or PEGDM 750 with poly (ethylene glycol) monofunctional methacrylate of mass 530 g. mol- ¹ have also been associated with the fibrin gel in order to vary the crosslinking density of the POE network, a total concentration of poly (ethylene oxide) monomer of 100 mg / ml is maintained. do not exhibit acceptable mechanical strength were obtained.

List of references

[1] Ruszczak, Z.. (2003) "Effect of collagen matrices on dermal wound healing". Advanced Drug Delivery Reviews 55: 1595-1611 [2] Nguyen, K. T. and J. L. West (2002). "Photopolymerizable hydrogels for tissue engineering applications." Biomaterials 23: 4307-4314.

[3] Schmedlen, R.H., J.L. Masters, et al. (2002). "Photocrosslinkable polyvinyl alcohol hydrogels that can be modified with cell adhesion peptides for use in tissue engineering." Biomaterials 23: 4325-4332. [4] From Gennes, P. -G. (1979). Scalinq Concepts in Polvmer Phvsics. London, Cornell University Press.

[5] Verderio E.A.M., Johnson, T., Griffin, M. (2004) "Tissue transglutaminase in normal and abnormal wound healing". Amino Acids 26: 387-404 [6] W. Bensaid, Triffitt JT, Blanchat, C. Oudina, K. Sedel, L., Small, H. (2003) "A biodegradable fibrin scaffold for mesenchymal stem cell transplantation", Biomaterials 24: 2497-2502.

[7] M.Fontanille, Y.Gnanou "chemistry and physico-chemistry of polymers" Dunod Edition, (2002) ISBN 2-10-049493-7 page: 410-413.

Claims

A process for preparing a material in the form of an interpenetrating polymer network (RIP) combining a physical fibrin gel and a polyethylene glycol (PEG) network, comprising the following steps: i) preparing a mixture by introducing into a buffer

a solution of fibrinogen,

monomers of formula (I):

Xr (CH ₂ -CH ₂ -O) _n -X ₂ wherein X and X ₂ are the same or different chemical moieties selected from the group consisting of vinyl, acrylate, methacrylate or allyl and n is a number between 1 and 140,

optionally monomers of formula (II) chosen from polyethylene glycol derivatives bearing a group chosen from the group comprising -CH ₃ , -NH ₂ , -OH, -CH ₂ CH ₃ , vinyl, acrylate, methacrylate or allyl; acrylate, methacrylate and styrene derivatives selected from the group consisting of hydroxyethyl methacrylate, hydroxyethyl acrylate, vinyl acetate, N-vinyl pyrrolidone, N-vinyl pyridine, acrylonitrile, acrylic acid , sodium styrene sulfonate, (vinylbenzyl) trimethylammonium bromide,

(vinylbenzyl) trimethylammonium, trimethylvinyloxycarbonylmethylammonium chloride, trimethylvinyloxycarbonylmethylammonium bromide, triethyl-2-vinyloxycarbonylethylammonium bromide, allyloxycarbonylmethyltrimethylammonium bromide, polyaluminum (ethylene glycol) methacrylate, poly (ethylene glycol) acrylate, - a polymerization initiator; ii) preparing a reaction mixture by adding a thrombin solution to the mixture prepared in i); iii) incubating the reaction mixture obtained in ii) at a temperature between 20 and 40 ⁰ C; iv) polymerization and crosslinking of the monomers of formula (I) with optionally the monomers of formula (II).

2. The method of claim 1, wherein the polyethylene glycol polymer (PEG) contains between 1 and 100% by weight of monomers of formula (I) and between 99 and 0% by weight of monomers of formula (II).

3. Method according to one of claims 1 or 2, wherein the monomers of formula (I) are those in which Xi and X ₂ , identical or different, selected from the group comprising methacrylates or acrylates and n is a number included between 2 and 20.

The method of any one of claims 1 to 3, wherein the polymerization initiator is a photopolymerization initiator.

The process according to claim 4, wherein the photopolymerization initiator is selected from the group consisting of Irgacures (2959, 184, 651, 819), 2-hydroxy-4- (2-hydroxyethoxy) -2-methylpropiophenone, benzophenone, 2,4-dimethylbenzophenone, benzoin, ionic benzophenones selected from the group comprising 4-trimethylmethylammonium benzophenone chloride, the sodium salt of 4-sulfomethylbenzyl, aromatic ketones and aldehydes including benzaldehyde, acetophenone, biacetyl, parachlorobenzophenone, ferrulic acid.

6. Process according to any one of claims 1 to 3, wherein the polymerization initiator is a thermal initiator.

The method of claim 6, wherein the thermal initiator is selected from the group consisting of peroxides, diazo compounds or persulfates.

The process of any one of claims 1 to 7 wherein in step iii) the incubation temperature is 37 ° C.

9. Process according to any one of claims 1 to 5 and 8, wherein in step iv), the polymerization is carried out under UV / visible radiation at a wavelength between 190 and 800 nm.

The process according to claim 9, wherein in step iv), the polymerization is carried out for 1 to 10 hours.

The process of any one of claims 1 to 3 and 6 to 8, wherein the polymerization takes place at the same time as step (iii).

12. Process according to any one of claims 1 to 3 and 6 to 8, wherein the polymerization takes place after step (iv), at a temperature between 25 and 60 ⁰ C.

13. The method according to any one of claims 1 to 12, wherein after step (iv), the method of the invention further comprises a step of enzymatic crosslinking of the physical fibrin gel.

The method of claim 13, wherein the enzyme used is transglutaminase.

15. Material in the form of an interpenetrating network of polymers (RIP) combining a physical fibrin gel and a polyethylene glycol (PEG) network obtainable by the method according to any one of claims 1 to 14.

16. The material of claim 15, wherein the polyethylene glycol (PEG) network contains between 1 and 100% by weight of monomers of formula (I) and between 99 and 0% of monomers of formula (II).

17. Material according to one of claims 15 or 16, wherein the monomers of formula (I) are those in which Xi and X ₂ , are identical or different chemical groups selected from the group comprising methacrylates or acrylates and n is a number between 2 and 20.

18. An interpenetrating polymer network (RIP) material according to any one of claims 15 to 17, comprising: a) from 1 to 50% by dry weight of fibrin; b) from 50 to 99% by dry weight of a polyethylene glycol (PEG) network.

19. Material according to any one of claims 15 to 18, having a glass transition temperature (Tg) of between -100 and +100 ⁰ C.

20. Material according to any one of claims 15 to 19, having a shear modulus (measured in shear mode G ') of between 100 Pa and 10 MPa in the hydrated state at 37 ° C and a modulus of conservation (measured in voltage mode E ') of between 0.01 and 3000 MPa in the dry state.

21. Use of a material according to any one of claims 15 to 20 or obtainable by the method according to any one of claims 1 to 14, such as:

- dressing for wounds,

- surgical dressing,

a device for delivering therapeutic agents; a coating for medical devices selected from the group comprising stents, heart valves, catheters, vascular prosthetic filters,

support of active molecules selected from the group comprising growth factors, antibiotics, bactericidal, bacteriostatic or enzymes,

or for eukaryotic cell cultures.