Dry substance hydratable in an aqueous gel containing particles of dispersed polymers, its preparation process and its biological application

The present invention relates to a hydratable dry matter in an aqueous gel containing dispersed polymer particles, its preparation process and its application in biology (diagnostic tests, cell culture, affinity chromatography, etc.).

European application No. 87.786 describes beads comprising a matrix of a hydrated agarose gel containing microspheres or powder of a polyaldehyde, for example polyacrolein; these beads can be used in various applications such as affinity chromatography, hemoperfusion, ion exchange resins, diagnostic tests ...

The drawbacks of such products lie in the problems encountered during their implementation (difficulty in synthesizing polyacroleins and toxicity of acrolein monomer); moreover, these beads are in the wet gel state which is difficult to handle.

The French patent published under No. 2,297,879 describes the preparation of rehydratable dried plates containing agarose or agar, usable in techniques such as immunodiffusion, electrophoresis, etc .; the described method consists in forming on a support an aqueous gel agarose or agar film containing a water-soluble acrylamide polymer and then drying the gel film.

These plates have the advantage of being able to be kept for an extended time and of being able to be rehydrated at the desired time; on the other hand, they can only be used in a limited field of application.

It is also known to fix proteins on polymer particles in aqueous dispersion, by absorption when it is a question of polystyrene particles for example, or by covalence when it is about polymers having reactive groups. The advantage presented by latexes results in the large specific surface developed by the particles and in their wide range of available chemical functions. Their drawback, on the other hand, lies in their colloidal instability, in particular with respect to electrolytes, and in the difficulty of recovering the particles on which the substances to be isolated are fixed.

The Applicant has found a dry matter consisting of a matrix capable of forming a porous gel in the presence of water, a matrix in which are dispersed particles of polymer derived from a water-immiscible monomer; this product has the advantage of being easy to handle and of being susceptible to multiple applications.

The hydratable dry matter in an aqueous gel which is the subject of the invention is characterized in that it comprises:<ul id="ul0001" list-style="none"><li>- a matrix consisting of a macromolecular substance A capable of forming a porous aqueous gel when it is in the presence of water.</li><li>- a water-soluble linear polymer B</li><li>- a plasticizer for said macromolecular substance A</li><li>- and, dispersed in said matrix, particles based on a polymer C derived from at least one water-immiscible monomer, having a glass transition temperature above 30_°C (preferably greater than 60_°C) and possibly having reactive functional groups.</li></ul>

The hydratable dry matter which is the subject of the invention can be in any form: films, plates, sticks, pellets, balls, etc.

The macromolecular substance A constituting the matrix can be chosen from polysaccharides, proteins, etc., the aqueous solutions of which are capable of forming a gel after cooling to a temperature of 30 to 80 ° C.

By way of example, mention may be made of polygalactoses such as agarose or agar, pectins, proteins such as gelatin, collagen.

The water-soluble linear polymer B present in the product of the invention can be chosen from water-soluble polymers whose viscosity at 22_°C in aqueous solution at 5% by weight is less than 17,000 centipoise (preferably less than 6,000 centipoise).

By way of example, mention may be made of polyethylene glycols, polyvinyl alcohol, poly (vinylpyrrolidone), hydroxyalkylcelluloses, carboxyalkylcelluloses and very particularly linear polyacrylamides.

The term “polyacrylamides” is understood to mean both linear homopolymers of acrylamide and linear copolymers of acrylamide and of at least one other comonomer of the vinylpyrrolidone type, acrylic or methacrylic ester of the monomethacrylate type of ethylene glycol or monoacrylate of ethylene glycol.

The quantity of water-soluble linear polymer B present in the hydratable dry matter corresponds to approximately 2 to 10 times the weight of the matrix.

Among the plasticizers of the macromolecular substance A mention may be made of polyols such as glycols, glycerol, sorbitol, polyethylene glycols of average molecular weight by weight less than 400.

The amount of plasticizer corresponds to approximately 1 to 3 times the weight of the matrix.

The particles based on polymer C dispersed in the matrix can have a particle size of the order of 0.05 to 20 μrn and preferably from 0.1 to 3 μm.

The weight of particles used corresponds to approximately 0.1 to 20 times (preferably approximately 1 to 10 times) the weight of the matrix.

Said polymer C is derived from at least one water-immiscible unsaturated monomer.

The term "water-immiscible monomer" means the monomers having a solubility in water of less than 5% by weight.

Among these we can cite:<ul id="ul0002" list-style="none"><li>- vinyl aromatic monomers (styrene, vinyl toluene ...)</li><li>- alkyl esters of unsaturated ap acids (methyl, ethyl acrylates and methacrylates ...)</li><li>- esters of unsaturated carboxylic acids (vinyl acetate) ...)</li><li>- vinyl chloride; vinylidene chloride</li><li>- dienes (butadiene ...)</li><li>- those with nitrile functions (acrylonitrile ...)</li><li>- siloxanes.</li></ul>

A first variant of constitution of the product which is the subject of the invention consists in that the polymer C is a copolymer derived from at least one monomer immiscible with water and of a small amount not exceeding 10% in weight, relative to the total weight of monomers (preferably not more than 4% by weight) of one or more comonomers carrying ionogenic or reactive groups such as - SO₃H, - OSO₃H, - N⁺R₃, - COOH, OH, NH₂, - NR₂, <chemistry id="chem0001" num="0001"><img file="EP0242275B1_D0001.tif" /></chemistry>- 0 CH₂CI, CONH₂ ..., R representing a C 1 alkyl radical₁ - VS₄, preferably in Ci - C₂

By way of example, we can cite:<ul id="ul0003" list-style="none"><li>- vinylbenzene sulfonate, sulfoalkyl esters of unsaturated acids (2-sulfoethylmethacrylate ...).</li><li>- unsaturated carboxylic acids (acrylic, methacrylic, maleic, itaconic ...)</li><li>- hydroxyalkylacrylates or methacrylates (hydroxyethyl acrylate, hydroxypropyl ...)</li><li>- aminoalkyl esters of unsaturated acids (2-amino-ethyl methacrylate ...)</li><li>- acrylamide</li><li>- vinylbenzyl chloride</li><li>- glycidyl methacrylate.</li></ul>

A second variant of constitution of the product which is the subject of the invention consists in that the polymer C is an alkali-soluble or acid-soluble copolymer, that is to say capable of being dissolved in the form of an aqueous solution of copolymer when the pH is made alkaline or acidic.

These copolymers are derived from at least one water-immiscible monomer and from 4 to 90% by weight relative to the total weight of monomers (preferably from 10 to 50%) from at least one aniogenic comonomer in the case of alkali-soluble or causative copolymers in the case of acid-soluble copolymers. The rate of comonomer is of course a function of the hydrophilia thereof.

By way of example of aniogenic comonomer, mention may be made of unsaturated carboxylic acids such as itaconic, maleic, fumaric acids, etc.

The pH of solubilization of the alkali-soluble copolymers thus formed is generally of the order of 6 to 11.

By way of example of a catiogenic comonomer, there may be mentioned:<ul id="ul0004" list-style="none"><li>1) The N (ωdialkylaminoalkyl) amides of unsaturated carboxylic acids of formula:<chemistry id="chem0002" num="0002"><img file="EP0242275B1_D0002.tif" /></chemistry>or : . Ri represents a hydrogen atom or a C 1 alkyl group_l-VS₄, and preferably in C₁-VS₂. R₂ represents an alkylene group C₁-VS₁₂, and preferably C₁-VS₈. <ul id="ul0005" list-style="none"><li>. R₃ and R ' ₃represent Ci-C alkyl groups₆, and preferably in C₁-VS₄, phenyls optionally substituted with a C1-C6 alkyl radical. Meet this formula dimethylaminomethyl-acrylamide or methacrylamide, dimethylamino-ethyl-acrylamide or -methacrylamide ...</li></ul></li><li>2) The unsaturated aminoesters of formula:<chemistry id="chem0003" num="0003"><img file="EP0242275B1_D0003.tif" /></chemistry>or :<ul id="ul0006" list-style="none"><li>. R '₁ represents a C alkyl group₁-VS₅ and preferably in C₁-VS₂,</li><li>. R '₂ represents a linear or branched alkylene group containing at least 2 carbon atoms and preferably at C₂-VS₁₂, especially in C₂-VS_B,</li><li>. R "₃ and R "₃ are the same or different and represent a C alkyl group₁-VS₆, and preferably C₁-VS₄, optionally substituted by a hydroxy radical, a phenyl group optionally substituted by a C 1 alkyl radical₁-VS₉, the total number of carbon atoms contained in the radicals R '₂, R "s and R '"₃ must be greater than 8 preferably greater than or equal to 10.</li></ul></li></ul>

This formula corresponds to diterbutylaminoethyl methacrylate, diterbutylaminopropyl methacrylate, dipentylaminoethyl methacrylate.

The pH of solubilization of the acid-soluble copolymers thus formed is less than 8 and preferably less than 5.

A particular case of this second variant of constitution consists in that the polymer C consisting of an alkali-soluble copolymer has a weight average molecular weight of less than 100,000 and preferably less than 50,000.

A third variant of constitution of the product which is the subject of the invention consists in that the polymer C is "sensitized", which means that active substances such as antibodies, antigens,. drugs, enzymes ... are immobilized on the particles of polymer C (the term "polymer C" corresponds to the definition given above and includes the two variants developed above and very particularly the alkali-soluble and acid-soluble polymers ).

Another alternative form of hydratable dry matter which is the subject of the invention consists in that the polymer particles C included in the matrix are magnetizable polymer particles.

Said magnetizable particles contain from 0.5 to 50% by weight (preferably from 0.5 to 35% and very particularly from 0.7 to 20%) of a magnetic charge whose size is less than 1 μrn and preferably between 0.002-0.05 µm; the magnetic charge is obviously fine enough to be able to be included in the polymer particles.

This magnetic charge can be constituted for example by:<ul id="ul0007" list-style="none"><li>. metals or their alloys such as: iron, iron-silicon, nickel, cobalt, or their alloys with molybdenum, chromium, copper, vanadium, manganese, aluminum, titanium;</li><li>. iron oxides: Fe₃0₄ or y-Fe₂0₃ pure or in combination or in mixture with other oxides such as cobalt, manganese, zinc, barium, rare earth oxides;</li><li>. chromium dioxide.</li></ul>

The hydratable dry matter which is the subject of the invention can be obtained by carrying out the following steps:<ul id="ul0008" list-style="none"><li>1) mixture:<ul id="ul0009" list-style="none"><li>. of an aqueous solution of a macromolecular substance A capable of forming after cooling to a temperature of 30 to 80_°C a porous aqueous gel when said substance is in the presence of water, at a concentration corresponding to the formation of said gel at a temperature of 30 to 80_°VS</li><li>. of a water-soluble linear polymer B</li><li>. a plasticizer for said macromolecular substance A. and a latex of a polymer C derived from at least one water-immiscible monomer, having a glass transition temperature greater than 30_°C (preferably greater than 60_°C) and possibly having reactive functional groups.</li></ul></li><li>2) cooling to a temperature below that of gelation of the aqueous solution of molecular substance A and shaping during the cooling of the aqueous gel obtained.</li><li>3) then drying at a temperature below said gelation temperature.</li></ul>

The raw materials used to carry out the mixing step are those already described above.

This mixing step is carried out at a temperature higher than that of forming an aqueous gel of macromolecular substance A; the concentration of macromolecular substance in the aqueous solution corresponds to that necessary for the formation of such a gel. Thus when the macromolecular substance is agarose, the mixing step is carried out using an aqueous solution at 0.5 to 2% by weight of agarose, at a temperature above 40_°C and generally less than or equal to 90_°vs.

The quantity of linear water-soluble polymer B used corresponds to approximately 2 to 10 times the weight of macromolecular substance A (dry).

The amount of plasticizer used corresponds to approximately 1 to 3 times the weight of macromolecular substance A (dry).

The water-soluble linear polymer B and the plasticizer can be used in the dissolved state either entirely in the aqueous solution of macromolecular substance A, or partly in this aqueous solution and partly in the polymer latex C.

The latex used contains 5 to 30% (preferably 10 to 15%) by weight of polymer particles.

The amount of latex used is equivalent to an amount of polymer C corresponding to 0.1 to 20 times (preferably approximately 1 to 10 times) the weight of macromolecular substance A (dry).

When, according to the third variant of constitution of the product which is the subject of the invention, particles carry immobilized active substances, said active substances can be introduced into the medium, either directly by using a latex of polymer particles C to which said substances have been fixed by physical absorption or chemically by covalence, either indirectly after encapsulation of the polymer particles C by the aqueous gel of macromolecular substance A, by migration of said active substances through the gel of macromolecular substance A then immobilization by physical absorption or by covalence.

The fixing of the biological molecules by covalence on the polymer particles can be carried out by coupling reaction, reaction involving the surface groups of the polymer particles and the functional groups of the biological molecule to be fixed.

This coupling reaction can be carried out according to well known methods, for example:<ul id="ul0010" list-style="none"><li>. by using coupling agents (such as glutaraldehyl, water-soluble carbodiimide)</li><li>. by activation of the functions of the polymer (for example by diazotization, by action of cyanogen bromide, hydrazine, etc.) then reaction with the molecule to be fixed.</li></ul>

When a hydratable and magnetizable dry matter is desired, the polymer latex used is a latex whose polymer particles are magnetizable.

Said latexes can be obtained according to known methods, for example according to the methods described in European patent N ° 38,730 or American patent N ° 4,157,323.

The operation of shaping the aqueous gel obtained by cooling is carried out according to a method depending on the shape desired for the final dry matter.

When the macromolecular substance used is agarose, this cooling step is favorably carried out at a temperature of 15 to 25_°vs.

The aqueous gel, once formed is dried under a stream of air, at a temperature close to that of the cooling step.

A gel in the form of a film can be obtained by pouring the mixture obtained in the first step onto a glass plate, allowing it to cool to transform the deposited liquid film into an aqueous gel film; the aqueous gel film can then be removed from the mold and dried as indicated above.

A particularly interesting mode of shaping the aqueous gel and drying is that described in the French patent published under No. 2,297,879.

The shaping process consists in pouring the mixture obtained in the first step onto a support consisting of a transparent thermo-plastic plate GEL-BOND sold by LKB placed on a horizontal glass plate, to allow to cool to form an aqueous gel. which adheres to the plate, to cover the film of aqueous gel with a sheet based on regenerated cellulose ("Cellophane"^@ marketed by Rhône-Poulenc) soaked in an aqueous solution of glycerol; the regenerated cellulose sheet is folded under the glass plate.

The whole is then dried at room temperature under a stream of air; the glass plate is finally detached from the plastic plate coated with hydratable dry matter.

The dry matter hydratable in an aqueous gel containing dispersed polymer particles forming the subject of the invention has the advantage of combining the positive properties of the matrix, namely being hydratable at the desired time in a porous aqueous gel under form of films, plates, balls ... easy to handle, to be compatible with aqueous media even very rich in electrolytes, to be permeable to high molecular weight proteins, to the positive properties of polymer particles derived from a water-immiscible monomer, namely to possess a surface high specific and controlled as well as a wide range of chemical functions available on the surface.

Said product is particularly interesting for its applications in biology; the porous nature of the gel of macromolecular substance A allows the proteins to reach the polymer particles C and to be fixed there by absorption or covalence. Many active substances such as antibodies, antigens, drugs, enzymes ... can thus be immobilized; depending on the nature of said immobilized active substance, a support can be obtained which can advantageously be used in immunoenzymatic or immunoradiological tests, in affinity chromatography, in extra-bodily purification systems, as an enzymatic catalyst in biotechnology, as device controlling drug delivery (drug release system), as a cell culture support.

The products of the invention in which the polymer particles C are alkali-soluble or acid-soluble with a low molecular mass (less than 100,000) and on which active substances are immobilized, are particularly advantageous in affinity chromatography; for example if such a product containing alkali-soluble polymer particles on which antibodies have been immobilized is put in the presence of a mixture of antigens, the antigen recognized by the antibody binds to the particle and remains attached to it after elution of the mixture of antigens; a simple increase in pH allows the polymer particles to dissolve and migrate through the porous gel, releasing the antigen-antibody complex formed.

The alkali-solubility (or acid-solubility) property of polymer C of any molecular mass can be exploited for the constitution of drug vectors where the active principle is released by simple modification of pH.

Another advantage of the hydratable dry matter in an aqueous gel containing dispersed polymer particles forming the subject of the invention, is that a judicious choice of the nature of the macromolecular substance A gives the possibility of selectively recovering the particles of polymer C onto which the substances to be isolated have been fixed, and this after destroying the macromolecular substance A after use, for example by enzymatic degradation when the macromolecular substance A is collagen.

The following examples are given for information only and cannot be considered as limiting the scope and spirit of the invention.

Example 1

Preparation of a rehydratable dry plate containing particles of polystyrene with a diameter of 0.8 microns.

8 g of linear polyacrylamide, the viscosity of which in 5% aqueous solution at 22 ° C. is close to 6000 centipoise, is dissolved at room temperature in 52 g of water containing 2 g of glycerol; this operation takes approximately 48 hours.

48 g of ESTAPOR @ K 080 latex (latex marketed by Rhône-Poulenc and consisting of 0.8 micron polystyrene monodispersed particles) containing 41% dry extract are then added to this preparation.

Furthermore, 2 g of agarose are dissolved in a boiling water bath in 100 ml of demineralized water containing 2 g of glycerol.

Both solutions are increased to 50_°C and then mixed, stirring slowly to avoid the formation of air bubbles.

The mixture obtained is poured onto a transparent plastic support GEL-BOND (marketed by LKB) placed on a horizontal glass plate, so as to obtain a layer of gel 0.8 mm thick.

The whole is cooled to room temperature; the aqueous gel adheres to the support.

The film of aqueous gel is then covered with a sheet of "Cellophane" @ (marketed by Rhône-Poulenc) previously soaked in a 2% glycerol aqueous glycerol solution. The edges of the "Cellophane" sheet are folded back under the glass plate and the whole is dried at room temperature under a stream of air for 15 hours.

The assembly consisting of the dried gel film of white appearance, the GEL-BOND support and the "Cellophane" sheet @ is then detached from the glass plate and stored at room temperature.

The rehydration of the dried gel film can be carried out by immersing the dried gel film stripped of the GEL-BOND support and the "Cellophane" sheet @ in water at room temperature; this water is renewed to remove the linear polyacrylamide; rehydration is accompanied by significant swelling of the gel without release of the polystyrene particles.

The specific surface area of ​​dry matter calculated per gram of dry matter is 4.4 m^2.

Example 2

Preparation of a rehydratable dry plate containing particles of 0.3 micrometer diameter carboxylated polystyrene.

The operations described in Example 1 are carried out using the following raw materials:<ul id="ul0011" list-style="none"><li>. Agarose 3 g</li><li>. glycerol 6 g (3g + 3g)</li><li>. polyacrylamide 6 g</li><li>. ESTAPOR® K1-030 Latex 40 g (the ESTAPOR K1-030 latex, sold by Rhône-Poulenc, is an aqueous dispersion of calibrated particles of carboxylated polystyrene; the measured diameter of the particles is 0.326 ± 0.010 micron; the rate of surface COOH functions is 273 microequivalents per gram of dry particles; the dry extract rate is 30%).</li></ul>

A rehydratable dry gel is thus obtained.

The specific surface area of ​​dry matter calculated per gram of dry matter is 13.2 m2.

The rate of carboxylic functions available per gram of dry matrix is ​​192 microequivalents.

Example 3

Preparation of a rehydratable dry plate containing particles of chlorobenzylated polystyrene 0.2 micron in diameter.

The operations described in Example 1 are carried out using the following raw materials:<ul id="ul0012" list-style="none"><li>. Agarose 1 g</li><li>. glycerol 2 g (1 g + 1 g)</li><li>. Polyvinyl alcohol 5 g (RHODOVIOL 4-125 marketed by Rhône-Poulenc)</li><li>. ESTAPOR K10-020 Latex 50 g (the ESTAPOR K10-020 latex, sold by Rhône-Poulenc, is an aqueous dispersion of calibrated particles of chlorobenzylated polystyrene; the measured diameter of the particles is 0.210 ± 0.006 micron; the rate of functions - 0 - Ch₂Cl is 200 microequivalents per gram of dry particles; the dry extract rate is 10%).</li></ul>

A rehydratable dry gel is thus obtained.

The specific surface of the dry matter calculated per gram of dry matter is 11 m2.

The rate of functions - 0 - CH₂CI available per gram of dry matrix is ​​77 microequivalents.

The presentation of the gel in its dry form makes it possible to keep the chlorobenzyl groups intact and to avoid their hydrolysis. This represents an important advantage since it has been found that the chlorobenzylated polystyrene latexes exhibit problems of storage stability; indeed during storage, the pH of such latexes which is 7-8 at the start can drop to 2 with release of chloride ions and formation of benzyl alcohol groups by partial hydrolysis of the chlorobenzyl groups present on the surface.

Example 4

Preparation of a rehydratable dry support containing magnetic carboxylated polystyrene particles.

The operations described in Example 1 are carried out using the following raw materials:<ul id="ul0013" list-style="none"><li>. Agarose 2 g</li><li>. sorbitol 5 g (2.5g + 2.5g)</li><li>. Polyvinylpyrrolidone 4 g (LUVISKOL K15 marketed by BASF)</li><li>. ESTAPOR MS1-070 / 25 Latex 150 g (the latex ESTAPOR MS1-070.25, marketed by Rhône-Poulenc is an aqueous dispersion of magnetic particles based on carboxylated polystyrene containing 25% by weight of magnetite Fe₃0₄ the average diameter is 0.7 micrometer; the rate of carboxylic functions is 40 microequivalents per gram of dry particles; the dry extract rate is 10%).</li></ul>

The dry plate obtained is cut into the form of squares with a side of 1 mm.

It is found that after rehydration the pieces are magnetizable using a laboratory magnet.

Example 5

Preparation of a rehydratable dry plate containing particles of polymer of alkali-soluble character.

The operations described in Example 1 are carried out using the following raw materials:<ul id="ul0014" list-style="none"><li>. Agarose 4 g</li><li>. ethylene glycol 4 g (2 g + 2 g)</li><li>. polyacrylamide 8 g</li><li>. alkali-soluble latex 20 g (the alkali-soluble latex is an aqueous dispersion of particles of a styrene / methacrylic acid / ethyl acrylate terpolymer of composition by weight 18/41/11; the particle size is of the order of 0.15 micrometers ; the dry extract rate is 38.5%; this terpolymer dissolves in water as soon as the pH is greater than 8; its viscosity in 10% aqueous solution is less than 150 centipoise at pH 9).</li></ul>

The dry plate obtained is rehydrated by soaking in slightly alkaline water (pH 9); it becomes translucent while retaining its cohesion and releases the polymer constituting particles into water.

Example 6

Preparation of a rehydratable dry plate containing polymer particles with an acid-soluble character.

The operations described in Example 1 are carried out using the following raw materials:<ul id="ul0015" list-style="none"><li>. Agarose 4 g</li><li>. ethylene glycol 4 g (2 g + 2 g)</li><li>. polyacrylamide 8 g</li><li>. acid-soluble latex 20 g (the acid-soluble latex is an aqueous dispersion of particles of a vinyl acetate / diethylaminoethyl acrylate copolymer with a 90/10 weight composition; the particle size is of the order of 0.15 microns; the rate of dry extract is 38%; this copolymer dissolves in water as soon as the pH is less than 3; its viscosity in 10% aqueous solution is less than 150 centipoise at pH 2).</li></ul>

The dry plate obtained is rehydrated by soaking in neutral water; the rehydrated plate becomes translucent while keeping its cohesion and releases into the water the polymer constituting the particles, if the pH is lowered below 2 by dilute hydrochloric acid.