WO1991008242A1 - Method for obtaining copolymers by grafting vinyl chain units, and graft copolymers thereby obtained - Google Patents

Abstract

Bioactive copolymers are prepared by radiochemically, chemically, or photochemically treating at least one compound A selected from a protein, a peptide, a protein or peptide derivative and a synthetic or natural material B, all at the same time, in the presence of one or more vinyl monomers, at least one homopolymerisation inhibitor and a cross-linkage agent, in order to graft the vinyl chain units onto A and B, and form a covalent association between them.

Description

 I

PROCESS FOR OBTAINING COPOLYMERS BY GRAFTING VINYL PATTERNS AND RESULTING GRAFT COPOLYMERS

The invention relates to a process for obtaining copolymers by grafting vinyl units and the resulting bioactive copolymers.

It relates more particularly to obtaining copolymers comprising at least one molecule having a biological activity, associated by irreversible bond to a macromolecular material, by means of grafted units of one or more vinyl monomers.

This type of copolymer has been studied to develop, for example, vascular prostheses or even active phases usable in affinity chromatography.

In these applications, it is a question of having, on the surface of a material, a bioactive molecule capable of interacting with the medium in which the material is found. According to the prior art, the covalent association of the bioactive molecule with the macromolecular support material is generally obtained by means of coupling agents. These agents most often request the reaction capacity of the -OH or -NH2 groups of the molecule to be bound. The probability for a large number of these sites belonging to the same molecule to participate in the association processes remains high, with the consequence of a parallel reduction in the conformational freedom of the molecule and a decrease in its biological activity. According to another route, some of the co-inventors of the present application have attempted to combine heparin by radiochemical grafting, using vinyl monomers on a polymer material constituted by a polyester, to form a vascular prosthesis (Baquey et al. Innov. Tech. Biol. Med. vol 2, n ° 4 1981, 379-389).

The operating conditions used made it possible to confer on the polyester anticoagulant properties, but these, associated with its defect of intrinsic impermeability, have not led to industrially usable products.

Developments in the inventors' work on the grafting technique using vinyl monomers have now made it possible to define operating conditions ensuring the establishment, between bioactive molecules and macromolecular materials, of covalent bonds, and making it possible to provide the material macro¬ molecular of biological properties of interest. The object of the invention is therefore to provide a process for obtaining bioactive copolymers ensuring better maintenance of the conformational freedom of the biological molecule and, thereby, of its biological activity.

It also aims to provide copolymers making it possible in particular to modulate the interactions of macromolecular materials with the biological media with which they are brought into contact.

The invention further relates to the biological applications of these copolymers for the preparation, for example, of vascular prostheses or of active phases usable in affinity chromatography.

The process for obtaining the bioactive copolymers of the invention is characterized in that at least one compound A chosen from a protein, a peptide, a protein or peptide derivative and a synthetic or natural material B is subjected simultaneously to radiochemical, chemical or photochemical treatment, in the presence of one or more vinyl monomers, of at least one homopolymerization inhibitor and of a crosslinking agent, so as to obtain the grafting of the units of vinyl origin, by irreversible association on A and B and the crosslinking of the vinyl motifs.

These arrangements make it possible to fix the product A on a macromolecular material B by means of polymeric chains of vinyl origin establishing bridges of variable length between A and B and by crosslinking between these bridges. This results on the one hand from a solid fixation of A on the macromolecular material B and a large

REPLACEMENT SHEET flexibility at the connection between A and B, crosslinking increasing the probability of establishing polymer bridges between the two partners A and B.

The choice of protein, peptide or protein or peptide derivative is made according to the specific application envisaged.

In general, use is made of a protein, a peptide or one of their derivatives endowed with a biological activity having a particular affinity for a given molecular or cellular ligand, for example a cellular receptor.

To make waterproof coatings of macromolecular textiles, with a view in particular to the development of vascular prostheses, collagen is advantageously used as protein A.

More particularly, collagen of bovine origin containing a preponderant proportion of type I collagen is used.

Other advantageous proteins have a pro-inhibitory action on coagulation proteases, such as antithrombin III (AT III), or pro-activator of fibrinolysis, an inhibitory action on platelet adhesion such as albumin, or alternatively a mediating action of fibrinolysis or a mediating action of cell adhesion and growth.

As pro-fibrinolytic factors, we will mention urokinase, tissue plasminogen activator (tpA) streptokinase.

It will be noted that the association of AT III with a macromolecular material allows in situ inhibition of the thrombin generated during the interaction between the material and the blood.

Proteins promoting cell attachment and / or intervening in the organization of the extracellular matrix of tissues such as fibronectin or laminin, used where appropriate in conjunction with other proteins, for example collagen, make it possible to have copolymers which promote cell attachment.

Still other proteins include elastin or growth factors. The proteins used according to the invention can be in admixture with other compounds, for example with proteoglycans and / or glycosaminoglycans.

The protein derivatives are for example glycoproteins or else lipoproteins. To have purification systems, it is also advantageous to use proteins which can be used in affinity chromatography techniques such as specific immunoglobulins or even antithrombin III. Among the peptides suitable for implementing the invention, mention will be made of those of interest in controlling cell adhesion and having or corresponding to particular sequences playing a role in the adhesion of cells to the extracellular matrix. In view of their properties, such peptides are advantageously incorporated into the structure of surfaces intended to be colonized by cells.

One such peptide sequence recognized by cell adhesion receptors is of the arg-gly-asp type.

This tripeptide sequence can be used alone, in a synthetic peptide or in a repeating sequence.

This sequence or any other sequence of interest can also be present in polymeric links.

The use according to the invention of a peptide or a polypeptide, or of any macromolecule containing a peptide sequence containing one or more tripeptide motifs as mentioned above makes it possible in particular to improve the behavior of the macromoleσular material B vis- with respect to fabrics.

The macromolecular material B is of synthetic or natural origin.

REPLACEMENT SHEET Synthetic materials which are particularly advantageous for the biological applications of these copolymers include polymers such as polyesters, polyurethanes, fiber-forming polymers. In a variant, the material B linked to A constitutes a coating on the surface of another type of material. The covalent bond therefore only involves the macromolecular network of the surface coating.

Natural macromolecular materials include, for example, cellulose, agarose, dextran, starch.

Preferably, the vinyl monomer is chosen from acrylic or methacrylic acid or acrylates and methacrylates of alkyl and hydroxyalkyl, in particular from C 1 to C 4, in particular methyl, derivatives of these compounds such as acrylamide or acrylonitrile . The vinyl monomer can be formed by multivinyl compounds.

The grafting is carried out by any suitable radiochemical, chemical or photochemical means.

According to a preferred embodiment of the invention, the grafting is carried out according to a radiochemical process.

The use of radioactive sources emitting gamma rays allows easy grafting.

The dose delivered is at least 1 kilogray. The dose rate is approximately 3 kilograys / hour.

Thanks to the addition of a crosslinking agent to the reaction medium, the grafting rates can be limited, which makes it possible to better maintain the conformational freedom of A and therefore its biological activity.

The crosslinking agent allows the branching and crosslinking of growing homopolymer chains. The probability of bridging between compounds A and B is thereby increased.

As suitable crosslinking agents, mention will be made of agents comprising at least two vinyl double bonds such as ethylene dimethacrylate or bis-acrylamide.

REPLACEMENT SHEET The grafting is preferably carried out in the liquid phase.

In one arrangement of this embodiment, the macromolecular material B is immersed in an aqueous solution containing the compound A, a vinyl or multivinyl monomer, the homopolymerization inhibitor and the crosslinking agent.

The proportions of reagents making it possible to obtain the satisfactory mechanical properties of the macromolecular material and an unchanged macroscopic appearance correspond to the use, as a percentage by weight, of the vinyl derivative at a rate of 15 to 25%, of the homopolymerization inhibitor to from 5 to 10% approximately, of the crosslinking agent at 0.001% to 0.25%, compound A representing approximately 30%, the remainder being water.

The homopolymerization inhibitor is a salt, for example a copper or iron salt, such as CuSO4 or Fe SO4.

Alternatively, one operates in the vapor phase. The grafting can be carried out photochemically or alternatively chemically, for example using redox systems such as the Ce ^{III +} / Ce ^{IV +} pair.

The invention also relates, as new products, to the graft copolymers as obtained by implementing the process defined above.

These copolymers are characterized in that they comprise a compound A chosen from a protein, a peptide, a protein derivative or a peptide derivative, associated by covalent bond to a macromolecular material B via polymeric links such as resulting grafting of vinyl type monomers or of a mixture of such monomers, forming a network by means of a vinyl crosslinking agent.

In these copolymers, A and B are as defined above. The polymer links come from vinyl or multivinyl monomers and their derivatives, as indicated above. Copolymers of the invention are ternary copolymers of protein A type / links of crosslinked vinyl origin / macromolecular material B.

Protein A is formed by a protein of a given type or comprises several proteins chosen according to the applications envisaged.

The macromolecular material can be formed of a single type of material or of composite materials or of complex structure in which only the surface serves as a support for protein A.

In other copolymers, compound A is a protein derivative, a peptide or a protein or peptide derivative as already defined.

Given their particularly satisfactory mechanical and sealing properties, the copoly¬ mers of the invention are specially suitable for the development of vascular prosthesis substitutes.

The establishment of cross-linked links between them, between the protein reticulum and, for example, a polyester constituting a vascular substitute ensures the maintenance of the protein component in vivo. The solidity of the links established between the protein matrix, for example collagen and polyester, and the alveolar morphology, appearing in the single figure, of this matrix favors the perennial installation of an endothelium following an implantation, or culture in vitro endothelial cells.

Other applications of the copolymers of the invention include the development of active phases which can be used in affinity chromatography, in particular for the purification of biological molecules or for carrying out cell sorting and including heparin Sepharose® and Protein A Sepharose represent particularly well-known examples. Thus, the binding of immunoglobulins to an appropriate material B makes it possible to selectively fix the antigens of a biological medium, specifically recognized by these immunoglobulins. ^•

REPLACEMENT SHEET Similarly, the fixing of ATIII on a support B. makes it possible to have a material for separating heparin or heparin fragments from a given medium or products having an activity modeled on that of heparin. The ability of compound A to be recognized by the molecules of the medium to be analyzed makes it possible to carry out separations with high yields.

Indeed, the molecules of A linked to the support B to which they bring a specific affinity for a given molecule, or a given cell type, retain their functional integrity better than when they are linked according to the chemical processes of the prior art.

To illustrate the invention, examples of preparation of graft copolymers (1) polyethylene terephthalate / links of vinyl / collagen origin or

(2) Cellophane ® / links of vinyl origin / collagen or albumin. In these examples, reference is made to the single figure which represents a photo of a view by scanning electron microscopy of a knitted sample of polyethylene terephthalate associated with collagen and acrylic acid according to the method of the invention. EXAMPLE 1 Study of the waterproofing of a grafted polyester prosthesis (polyethylene terephthalate / acrylic acid / collagen copolymer).

- Grafting protocol

. Preparation of polyester samples The prostheses are cut into samples, weighed, washed with boiling distilled water in a soxhlet apparatus until constant mass.

• Preparation of reagents

- Acrylic acid. The acrylic acid supplied by MERCK contains 200 ppm of hydroquinone as a polymerization inhibitor. This inhibitor can be removed by distillation of the monomer which is then stored under vacuum in ampoules at -80 ° C.

REPLACEMENT SHEET _g_

- Collagen

Collagen of bovine origin is presented in lyophilized form, supplied by ORGANOTECHNICS.

It is dissolved in a 0.5 mol / l glacial acetic acid solution at a concentration of 1.7%, then stored at +4 ^β C.

- Copper sulfate

A 10-2 mol / l aqueous solution of copper sulphate is produced. Added to the reaction medium, it makes it possible to avoid excessive homopolymerization of the monomer (s) in solution during the irradiation.

- Ethylene dimethacrylate

The crosslinking agent is stored at + 4 ° C. and used as it is at very low concentration. . Procedure

A grafting solution containing 15 to 30% of acrylic acid, 5 to 10% of a 10 ~ 2M solution of CuSO 4, 5H20, 40% of collagen solution, 0.01% to 0.25% of is used. crosslinking agent with 100% water. The polyester samples are placed in a glass enclosure containing the homogenized grafting solution. The contents of the cell are degassed by 4 successive cycles of freezing - pumping - thawing.

Freezing is carried out by immersion of the enclosure in liquid nitrogen (-196 ° C).

The vacuum is achieved using a vane pump.

The cell is thawed in a water bath at ^• 37 ° C. The number of cycles performed depends on the amount of solution to be degassed. The quality of degassing is assessed visually.

The cell containing the degassed solution and the samples is placed in an irradiator to undergo the range irradiation necessary for the polymerization of the various reagents. The ^samples are then recovered, washed in an aqueous solution of acetic acid to 1/500, for lh with stirring at room temperature. They are washed with distilled water for 3 days so as to remove the insufficiently bonded copolymer to the wall. When ^'the excess of copolymer is removed, the dentures were frozen, lyophilized and weighed. EXAMPLE 2 Preparation of a copolymer

Cellophane ¹ / acrylic acid / albumin or collagen.

By gamma irradiation until absorption of 2 kilograys (gamma of 661 keV of 137cesium), at a dose rate of 300,000 rad.h-1, of Cellophane ® pre-impregnated with a solution of albumin or collagen and exposed in the absence of oxygen in the saturated vapor of acrylic acid, one obtains solution of albumin or collagen and exposed in the absence of oxygen in the saturated vapor of acrylic acid ^, one obtains the irreversible association of the protein with Cellophane ®. The material obtained manifests a different biocompatibility than that of the starting cellophane, the difference observed depending on the protein used.

EXAMPLE 3 Preparation of a Dacron® / acrylic acid / collagen copolymer.

By gamma irradiation until absorption of 10 to 15 kilograys (gamma of 661 keV of 137cesium) at a dose rate of 300,000 rad.h- !, of polyethylene terephthalate (DACRON ^) knits prepregs with an aqueous solution of collagen and exposed in the absence of oxygen to the saturated vapor of acrylic acid, the irreversible association of collagen with polyester is obtained and the immediate wall sealing of the prostheses made from these modified knits.

In addition, advantageously, the affinity of the treated material, for the platelets on the one hand, and for fibrinogen on the other hand, is modified compared to that of the starting polyester.

STUDY OF THE STABILITY OF GRAFT COPOLYMERS

We report the results of a study carried out with human serum albumin (ASH) labeled with 125ι grafted with Cellophane © • . Cellophane ® samples are incubated in the presence of an albumin solution (200 mg / ml) containing albumin labeled with iodine 125.

A first series of samples (series A) is rinsed with the solvent buffer until a constant value of their residual radioactivity is obtained; then, the rinsing is continued with an albumin solution of the same concentration as the solution used during the incubation, but free of radioactive tracer. There is then a decrease in the residual radioactivity of the samples, proving an exchange of pre-adsorbed albumin molecules, and possibly labeled, with cold albumin molecules.

treatment described by the invention (Example 2). The samples are then rinsed with water and then with an albumin solution without radioactive tracer like the samples of series A. There is practically no reduction in their residual radioactivity, which leads to the conclusion that the irreversible association of

1 albumin with Cellophane ®.

The same type of experiment was carried out with the Cellophane ® system - collagen, and the polyester system - albumin, the results obtained each time confirming the establishment of an irreversible bond between the compound

A and material B.

EXAMPLE 4: BIOLOGICAL CHARACTERIZATION. Culture of human endothelial cells (CEH) in the presence of extracts of copolymers of the invention (collagen / acrylic acid / Dacron®).

Grafted samples according to the invention and ungrafted samples are sterilized with 25 kilogray gamma rays. The extracts of tested materials are prepared from samples immersed for 120 hours at 37 ° C in physiological saline (0.9% NaCl). The volume of serum used is such that the ratio of the surface area of the sample to this volume is equal to 5 cm2 / ml. The final concentration of the extract in the culture medium is 10% (vol / vol).

The CEH are isolated from umbilical cord veins according to the method described by Jaffe in Transplantation proceedings 1980 (Sept) XII, n ° 3 Suppl-1, 49-53.

On the third pass, the cells are inoculated into containers containing several wells at a starting density of 7.5 × 10 3 cells / cm 2. The cells are maintained at 37 "C under 5% C02 and the culture medium is renewed every day. At 37 ° C under 5% CO 2 and the culture medium is renewed every day _e.

The results obtained are reported on 4 series:

a control cultivated with a complete medium composed of M 199, 20% of fetal calf serum, Endothelial Cell Growth Supplement *, at a rate of 20 μg / ml and of heparin at a rate of 90 μg / ml,

- a positive control cultured with the same medium containing 6.4 μg of phenol / ml (which causes constant and reproducible cytotoxicity),

- a control test, containing the same medium and an extract of ungrafted material,

- a test containing the same medium and an extract of graft copolymer.

The biocompatibility of the copolymer is evaluated by cell attachment (after 3 to 6 hours), proliferation and the protein content of the cells.

REPLACEMENT SHEET At the end of the chosen incubation period, the medium is eliminated, the cells are detached with trypsin and counted.

To perform a quantitative determination of cellular proteins, the cells are rinsed and then lysed in distilled water using five freeze-thaw cycles.

The total protein content (in ug / 105 cells) is determined according to the technique known as Pierce BCA protein assay (Interchim, Paris). For specific biocompatibility, the intracellular presence of factor VIII (FVIII) is revealed by immunofluorescence in the controls and the series tested on the 6th day of proliferation.

A complete attachment of the cells is observed for all the series in 3 hours. On the 6th day, the cells are homogeneous. The series tested are morphologically similar to the control.

The study of proliferation kinetics has shown that the presence of graft copolymer extracts does not lead to inhibition.

The CEH express and maintain their characteristics, namely the synthesis of FVIII antigen, even in the presence of extracts of copolymer materials according to the invention.

EXAMPLE 5 Application of the method of the invention to the association of a protein in a chromatography phase.

The phase chosen is SEPHADEX ® G 25 (crosslinked Dextran) and the protein albumin.

Sephadéx ® (1 g) is swelled in an albumin solution (7.0 mg / ml) accompanied by albumin labeled with iodine 125 in physiological phosphate buffer (PBS)

- pH = 7.4), for 10 hours. 1 ml of acrylic acid, 1 ml of copper sulphate solution (5.10 ^-2 M) are added.

The final suspension is degassed under a vacuum of 5.102 torr, then irradiated until absorption of 1 Mrad at a dose rate of 0.3 Mrad.h-1 approximately. The Sephadex® gel is recovered by decantation and then extracted with a soxhlet using a 1: 1 mixture (methanol / water) for 5 days.

It is then rinsed on a column with 1000 ml of double-distilled water at a flow rate of 0.5 ml / min. He is finally freeze-dried.

The radioactivity measurements make it possible to calculate a retention rate of 1.03 mg / g of dry Sephadex®, which is 8 times more than the quantity retained when the Sephadex® exposed to the albumin solution is not irradiated and rinsed with 100 ml of water only (no hot extraction).

The irradiation in the presence of acrylic acid therefore leads to stabilization of the albumin in the Sephadex® gel.

The invention therefore provides graft copolymers of high stability, the structure of which makes it possible to take full advantage of the biological activity of the compound of protein or peptide type, which is of major interest in the various biological applications where these copolymers are found. appropriate.

Claims

1 / Process for obtaining bioactive copolymers, characterized in that at least one compound A chosen from a protein, a peptide, a protein or peptide derivative, and a synthetic or natural material B is subjected simultaneously to radiochemical treatment , chemical or photochemical, in the presence of one or more vinyl monomers, at least one homopolymerization inhibitor and a crosslinking agent, so as to obtain grafting of the units of vinyl origin, by irreversible association on A and B. 2 / A method according to claim 1, characterized in that compound A has a specific affinity for a given molecular or cellular ligand. 3 / A method according to claim 1 or 2, characterized in that the compound A is collagen. 4 / A method according to claim 1 or 2, characterized in that protein A has a pro-inhibitory action of the coagulation proteases, or pro-activator of fibrinolysis, an inhibitory action of platelet adhesion or a mediating action of the fibrinolysis or a mediating action of adhesion and cell growth. 5 / A method according to claim 1 or 2, characterized in that protein A is chosen from proteins promoting cell attachment such as fibronectin or laminin and ^' is used where appropriate with other proteins such as collagen , among elastin or growth factors, said protein A being used where appropriate in admixture with other compounds such as proteoglycans and / or glycosaminoglycans. 6 / A method according to claim 1 or 2, characterized in that protein A is a protein usable in affinity chromatography techniques, such as specific immunoglobulins and Antithrombin III. 7 / A method according to claim 1 or 2, characterized in that compound A is a peptide or a peptide derivative consisting of or comprising a sequence involved in cell adhesion, in particular a tripeptide sequence of the arg-gly-asp type . 8 / A method according to any one of claims 1 to 6, characterized in that the macromolecular material B is a synthetic material, in particular consisting of a polymer such as a polyester, a polyurethane or a polymer forming fibers or a material natural such as cellulose, agarose, dextran or starch. 9 / A method according to any one of claims 1 to 8, characterized in that the reaction medium is subjected to a radiochemical treatment, more particularly by irradiating with the aid of rays emitted by radioactive rays. 10 / A method according to any one of claims 1 to 9, characterized in that the grafting is carried out in the liquid phase, the macromolecular material B being advantageously immersed in an aqueous solution containing the compound A, a vinyl or multivinyl monomer, the homopolymerization inhibitor and crosslinking agent. 11 / Bioactive copolymers, characterized in that they comprise at least one compound A chosen from a protein, a peptide, a protein or peptide derivative, associated by covalent bond to a macromolecular material B, via polymeric links such as resulting from the grafting of vinyl monomers or of a mixture of such monomers, forming a network via a vinyl crosslinking agent. 12 / Copolymers according to claim 11, characterized in that A and B are as defined in any one of claims 1 to 8. 13 / Copolymers according to claim 12, characterized in that they are ternary copolymers comprising collagen and a polyester linked by bridges REPLACEMENT SHEET vinyl crosslinked to each other via a crosslinking agent. 14 / Bioactive copolymers as obtained by implementing the method according to one of claims 1 to 10. 15 / Vascular substitutes, characterized in that they are produced from the copolymers according to one of claims 11, 12 and 13. 16 / Vascular substitutes, characterized in that they are produced from the copolymers according to claim 13. 17 / Active phases usable in affinity chromatography, characterized in that they are produced from copolymers according to one of claims 11 to 14. REPLACEMENT SHEET