WO2001052979A1

WO2001052979A1 - Method for preparing stable and functionalised colloidal particles and resulting particulate reagent

Info

Publication number: WO2001052979A1
Application number: PCT/FR2001/000206
Authority: WO
Inventors: Abdelhamid Elaissari; Bernard Mandrand; Thierry Delair
Original assignee: Bio Merieux
Priority date: 2000-01-21
Filing date: 2001-01-22
Publication date: 2001-07-26
Also published as: FR2804046B1; FR2804046A1

Abstract

The invention concerns a method for preparing functionalised stable particles which consists in: (i) providing a colloidal dispersion of organic and/or inorganic particles in an aqueous medium, said particles having functional groups X capable of interacting with other functional groups, said functional groups X being selected among amine, hydroxyl, thiol, aldehyde, ester, anhydride, acid chloride, carbonate, carbamate, isocyanate and isothiocyanate groups or mixtures thereof; (ii) contacting said dispersion with a dissolved polymer bearing functional groups, capable of ionisation, identical or different, selected among amine, carboxylic acid, ester, anhydride, aldehyde, thiol, disulphide, α-halogenocarbonyl, sulphonic acid, isocyanate and isothiocyanate groups to form a mixture; and (iii) incubating said mixture in predetermined conditions of temperature, pH and incubation time, so that at least a fraction of said functional groups X reacts with at least a fraction of the functional groups Z and/or Z' to form covalent bonds. The invention also concerns a particulate reagent having affinity for biological compounds.

Description

PROCESS FOR THE PREPARATION OF STABLE AND FUNCTIONALIZED COLLOIDAL PARTICLES AND OBTAINED PARTICULATE REAGENT

The stability of colloidal particles depends on the interaction of different interparticle attractive and repelling forces. The attractive forces are the van der Waals forces and the repulsive forces are either electrostatic or steric in nature.

The role of electrostatic effects in colloidal stability has been described in the DLVO theory (Derjagun-Landau-Verwey-Overbeek). When the electrostatic effects are canceled, for example by adding salt, the particles, whose stability was ensured by these repulsive forces, flocculate.

In order to maintain colloidal stability in a medium rich in salt, the particles must have steric stabilization (Jayachandran et al., J.M.S. - Pure Appl. Chem. A35 (12) 1971-1986, 1998). In this article, the authors have attached "arms" of polyoxyethylene to latex particles carrying amine functions on the surface. This method makes it possible to obtain stable particles, by steric stabilization, but the functional groups are consumed by the grafting of the arms and it is then no longer possible to graft molecules, for example biological molecules, onto the particles thus obtained.

The present inventors have now found a process which makes it possible to obtain colloidal particles, stable and functionalized, even in a medium rich in salt, by electrostatic and steric stabilization and which have many functional groups available for the covalent grafting or not of compounds organic.

According to this method, there is a colloidal, stable and isodisperse dispersion of organic and / or inorganic particles in an aqueous medium, said particles having functional groups X capable of interacting with other functional groups, said functional groups X being chosen from amine, hydroxyl, thiol, aldehyde, ester, anhydride, acid chloride, carbonate, carbamate, isocyanate and isothiocyanate groups or their mixtures, said dispersion is brought into contact with a solution of a polymer carrying functional groups ionizable, Z and Z ', identical or different, chosen from the amine, carboxylic acid, ester, anhydride, aldehyde, thiol, disulfide, α-halocarbonyl, sulfonic acid, isocyanate and isothiocyanate groups to constitute a mixture, and said mixture is incubated under conditions predetermined by a person skilled in the art who knows how to adjust the temperature, the pH and the incubation time under the conditions of the experiment, so that the groups Z, Z ′ partially react with the X groups of the heart By partial reaction of the functional groups Z, Z 'with X, it is understood that groups Z and / or Z' must remain available. Indeed, during the implementation of these particles, for example, in the immobilization of biological compounds, all or at least part of these free Z and / or Z groups will react by covalent interaction and / or by electrostatic interaction with the ionic groups of the biological compounds to be immobilized.

According to a preferred incubation step during the process for preparing the particles, the temperature is between 15 and 60 ° C., advantageously between 20 and 35 ° C., the incubation time is between 5 minutes and 24 hours, from preferably between 10 minutes and 3 hours, and the pH is between 6.5 and 7.5.

Table 1 below summarizes the complementarities between the different functional groups X, Z and Z '.

01/52979

Table 1

According to an object of the invention, the colloidal dispersion is a dispersion of organic particles consisting of at least one organic polymer chosen from at least one homopolymer or a copolymer or their mixtures, resulting from the polymerization of at least one monomer chosen from acrylamide and acrylate monomers, in particular N-alkylacrylamide and N, N-dialkylacrylamide, such as N-isopropylacrylamide, N-methylacrylamide, N-ethylmethacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N, N-diethylacrylamide, N-methyl-N-isopropylacrylamide, N-methyl-Nn-propylacrylamide; alkyl acrylates and methacrylates in which the alkyl group comprises from 3 to 20 carbon atoms; styrene, methylstyrene, ethylstyrene, tert-butylstyrene, chloromethylstyrene, vinyltoluene; their derivatives and the copolymers of these monomers with one another and / or with other comonomers.

According to another embodiment of the process of the invention, the dispersion is a dispersion of organic and inorganic particles, said organic particles being constituted by at least one organic polymer chosen from at least one homopolymer or copolymer or their mixtures, resulting from the polymerization of at least one monomer chosen from acrylamide and acrylate monomers, in particular the N -alkylacrylamide and NN-dialkylacrylamide, such as N-isopropylacrylamide, N-methylacrylamide, N-ethylmethacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylmethacrylamide, N-cyclopropylamide , N-methyl-N-isopropylacrylamide, N-methyl-Nn-propylacrylamide; alkyl acrylates and methacrylates in which the alkyl group comprises from 3 to 20 carbon atoms; styrene, methylstyrene, ethylstyrene, tert-butylstyrene, chloromethylstyrene; their derivatives and the copolymers of these monomers with one another; and said inorganic particles being chosen from particles of metal oxides, iron, titanium, cobalt, zinc, copper, manganese, nickel; magnetite; hematite, ferrites, such as manganese, nickel, manganese-zinc ferrites; cobalt, nickel alloys; zeolites; talc; clays such as bentonite and kaolin; alumina; ' silica, graphite; carbon black or other inorganic materials. Furthermore, in the process of the invention, the envelope polymer is chosen from at least one homopolymer or a copolymer chosen from homopolymers or copolymers:

- resulting from the polymerization of at least one monomer chosen from monomers derived from acrylamide or from methacrylamide; acrylic acid, methacrylic acid; acrylate and methacrylate derivatives; allylamine; styrenic derivatives; on the condition if it is a homopolymer that it contains ionizable functional groups; in particular the copolymers or homoplymers of maleic anhydride and the homopolymers or copolymers of acryloxysuccinimide, - polysaccharides, such as chitosan and polyactactic acid,

- polypeptides, such as polylysine and polyarginine,

- linear or branched polyethyleneimine, and

- dendrimers; in particular, poly (maleic anhydride methyl vinyl ether), poly (N-vinylmorpholine-N-acryloxysuccinimide) or poly (N-vinylpirrolidone-N-acryloxysuccinimide).

The invention also relates to a particulate reagent having an affinity for biological compounds comprising or consisting of organic and / or inorganic colloidal particles, stable and functionalized, comprising a core and an envelope. The core is essentially solid, organic and / or inorganic, and has functional groups X chosen from amine, hydroxyl, thiol, aldehyde, ester, anhydride, acid chloride, carbonate carbamate, isocyanate, isothiocyanate or mixtures thereof, including at least one fraction reacted with other functional groups of the envelope, and the envelope consists of a polymer carrying functional groups, ionizable, Z and Z ', identical or different, chosen from amine, acid groups carboxylic, ester, anhydride, aldehyde, thiol, disulfide, α-halocarbonyl, sulfonic acid, isocyanate and isothiocyanate, which partially reacted with the functional groups X of the heart.

The term “biological compound” is intended to mean in particular proteins, including holoproteins and heteroproteins or their fragments, that is to say, lipoproteins, glycoproteins, hemoproteins, phosphoproteins, flavoproteins, metalloproteins, polypeptides, antigens, immunogens, antibodies, enzymes. We also understand the associations of proteins and nucleic acids such as complex nucleoprotein structures such as chromosomes, histones, but also viruses, bacteria, fungi. Also included in biological compounds, nucleic acids and their fragments, are oligonucleotides.

In one embodiment of the invention, the core is organic and comprises at least one organic polymer chosen from at least one homopolymer or a copolymer or their mixtures, resulting from the polymerization of at least one monomer chosen from monomers of acrylamide and acrylate, in particular N-alkylacrylamide and N, N-dialkylacrylamide, such as N-isopropylacrylamide, N-methylacrylamide, N-ethylmethacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylm , N-cyclopropylacrylamide, N, N- diethylacrylamide, M-methyl-N-isopropylacrylamide, N-methyl-Nn- propylacrylamide; alkyl acrylates and methacrylates in which the alkyl group comprises from 3 to 20 carbon atoms; styrene, methylstyrene, ethylstyrene, tert-butyl-styrene, chloromethylstyrene vinyltoluene; their derivatives and the copolymers of these monomers ente them and / or with other comonomers.

In another embodiment of the invention, the core is organic and inorganic and comprises: at least one organic polymer chosen from at least one homopolymer or a copolymer or their mixtures, resulting from the polymerization of at least one monomer chosen from acrylamide and acrylate monomers, in particular N-alkylacrylamide and N, N-dialkylacrylamide, such as N-isopropylacrylamide, N-methylacrylamide, N-ethylmethacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N, N-diethylacrylamide, N-methyl-N-isopropylacrylamide, N-methyl-Nn-propylacrylamide; alkyl acrylates and methacrylates in which the alkyl group comprises from 3 to 20 carbon atoms; styrene, methylstyrene, - ethylstyrene, tert-butyl-styrene, chloromethylstyrene vinyltoluene; their derivatives and the copolymers of these monomers ente them and / or with other comonomers; and inorganic particles chosen from particles of metal oxides, iron, titanium, cobalt, zinc, copper, manganese, nickel; magnetite; hematite, ferrites, such as manganese, nickel, manganese-zinc ferrites; cobalt, nickel alloys; zeolites; talc; clays such as bentonite and kaolin; alumina; silica; graphite; carbon black or other inorganic materials

The envelope polymer is chosen from at least one hydrophilic homopolymer or copolymer chosen from homopolymers or copolymers: - resulting from the polymerization of at least one monomer chosen from monomers derived from acrylamide or from methacrylamide; acrylic acid, methacrylic acid; acrylate and methacrylate derivatives; allylamine; styrenic derivatives; on the condition, if it is a homopolymer, that it contains ionizable functional groups; in particular the copolymers or homoplymers of maleic anhydride and the homopolymers or copolymers of acryloxysuccinimide, - polysaccharides, such as chitosan and poly galacturonic acid,

- polypeptides, such as polylysine and polyarginine,

- linear or branched polyethyleneimine, and - dendrimers; in particular poly (maleic vinyl ether anhydride) poly (N-vinylmorpholine-N-acryloxysuccinimide) or poly (N-vinylpirrolidone-N-acryloxysuccinimide).

The colloidal particles of the invention, and in particular the reagent obtained from these particles, are in particular used for:

(i) grafting of molecules via their available reactive functions, in particular the grafting of biological molecules, such as nucleic acids, fragments of nucleic acids, oligonucleotides, peptides, proteins, fragments of proteins (the term peptides, proteins and fragments of proteins including proteins according to their usual definition, ie proteins produced by the expression of a gene, antigens and immunogens, but also antibodies, fragments' d 'antibodies-, enzymes); the grafting of non-biological molecules, such as markers, for example fluorescent, luminescent molecules or radioisotopic markers; drug substances; pigments; mineral fillers,

(ii) the complexation of inorganic products, in particular of metals, such as heavy metals in effluents,

(iii) assisted flocculation, (iv) enhanced recovery of hydrocarbons, in particular heavy hydrocarbons.

Thus, the particles of the invention can be used in the fields of analysis, in particular biological or chemical analysis; in methods of extraction or isolation and / or purification and / or concentration of biological or chemical molecules and more specifically in diagnostic tests, methods of extraction, purification and concentration of proteins and nucleic acids, methods of purifying and screening drug substances or compounds for vaccine use and depollution methods. The appended figure represents the measurements of electrophoretic mobility carried out on the latex. Before. Modification (represented by a solid line) and on conjugate 2 (represented by a broken line) of Example 4. The pH is presented on the abscissa and the electrophoretic mobility is represented on the ordinate.

Example 1: Obtaining magnetic carboxylic latexes.

The poly (maleic methyl vinyl ether anhydride) (AMVE) (Molar mass: 67 kDa) is dissolved in anhydrous dimethyl sulfoxide (DMSO) (2 g / l). 50 μl of this AMVE solution are diluted in 1 ml of phosphate buffer (pH 6.8; 10 mM) and then incubated for 10 minutes at 37 ° C. Then mixed 1 ml of a dispersion of amino magnetic latex (0.5% in water 1 times the critical micelle concentration (CMC) of Triton X-405) manufactured, for example according to the protocol described in the patent application PCT WO 99/35500, with 125 μl of the mixture (AMVE-DMSO-buffer) previously prepared. The mixture is incubated at 37 ° C for 3 hours. The particles can then be used as such or after a purification step, for example by magnetization or centrifugation.

Example 2: Grafting of oligonucleotides [H2N- (CH2) 6-dT14)].

0.14 mg of magnetic latex modified by grafting

12 of AMVE (in 10 mM phosphate buffer, pH 6.8) is mixed with 10 copies of oligonucleotides [H2N- (CH2) 6-dT14)] contained in milliQ water, 200 μg of an agent activation, N-ethyl carbodiimide hydrochloride (EDC) in the presence of 0.07% Triton X-405. The coupling is carried out in "batch", all the reagents being mixed at the same time, for 2 hours, at 37 ° C. Excess reagent is removed by successive washes with 10 mM phosphate buffer, pH 6.8.

The presence of the oligonucleotides is revealed by the ELOSA "Enzyme-Linked Oligosorbent Assay" technique (Katz JB et al., Am. J. Vet. Res. 1993 Dec; 54 (12): 2021-6 and François Mallet et al., Journal of Clinical Microbiology, June 1993, p1444-1449)).

Example 3: Synthesis of amino particles.

To an amount of 49 grams of deionized and degassed water under nitrogen, 4.5 grams of styrene and 0.5 grams of N-isopropylamide (NIPAM) (sold by the company Kodak) are added. The mixture is brought to 70 ° C., then 0.05 gram of 2.2 'hydrochloride is added. azobis (2 amidinopropane) (Wako). The radical polymerization reaction is allowed to proceed for 4 hours. A mixture consisting of 0.5 grams of NIPAM, 0.055 grams of aminopropyl methacrylamide hydrochloride (Kodak), 0.017 g of N, N 'methylenebisacrylamide (Aldrich) contained in 5 ml of water is then added. Then added to the reaction medium a solution of 0.025 gram of 2,2 'azobis (2 amidinopropane) (Wako) in 1 ml of water. The reaction is allowed to proceed for 18 hours.

The latex obtained has a solid content of 8.8% and the particle diameter is 358 nm at 20 ° C.

Example 4: functionalization of the amino latexes by AMVE.

A solution of AMVE polymer at 1 g / l in anhydrous DMSO is prepared. 250 μl of this solution are dissolved in 4.75 ml of phosphate buffer (pH 6.8; 10 mM). The whole is incubated for 10 minutes at approximately 37 ° C. At room temperature, 475 μl of dispersion of the latex prepared according to Example 3 is then added, at 0.5% of solid content. The mixture is incubated for 3 hours at 37 ° C. The dispersion can be used as it is or after a purification step by centrifugation.

The amount of AMVE can be varied by modifying the test sample of the polymer solution in DMSO, as shown in Table 2 below, which reports the variation in particle size as a function of the amount of polymer. added.

Table 2

#: means the particle diameter at 20 ° C and 0.001 M NaCI. 10

It should be noted that neither the DMSO nor the polymer, all of the anhydride functions of which have been hydrolysed before being brought into contact with the particles, cause the particle diameter to vary.

The electrophoretic mobility measurements carried out on the latex before modification and on conjugate 2, shown in the appended figure, show that the latex on which the polymer has reacted has a charge reversal, which demonstrates the presence of AMVE on the surface particles.

Example 5: Study of the stability of latex particles.

The colloidal stability of conjugate 2 and of a nonfunctionalized latex was carried out by following the evolution of the size of the particles as a function of the increase in salinity. An increase in size indicates flocculation of the particles. The measurements were carried out on the N4 device (Coultronics). The results are presented in Table 3.

Table 3

No flocculation is observed with the conjugate 2 which has fixed the AMVE polymer while the latex carrying amine functions and mainly electrostatically stabilized has started to flocculate.

Claims

1. Process for the preparation of functionalized stable colloidal particles according to which, - a colloidal, stable and isodisperse dispersion is available, of organic and / or inorganic particles in an aqueous medium, said particles having functional groups X capable of interacting with other functional groups, said functional groups X being chosen from amine, hydroxyl, thiol, aldehyde, ester, anhydride, acid chloride, carbonate and carbamate, isocyanate, isothiocyanate groups or mixtures thereof,

- said dispersion is brought into contact with a solution of a polymer carrying functional groups, ionizable, Z and Z ′, identical or different, chosen from amine, carboxylic acid, ester, anhydride, aldehyde, thiol, disulfide, α groups -halocarbonyl, sulfonic acid, isocyanate, isothiocyanate to form a mixture, and

- Said mixture is incubated under predetermined conditions of temperature, pH and incubation time, so that the functional groups Z and / or, Z 'partially react with the functional groups X,' for the formation of bonds covalent.

2. Method according to claim 1, characterized in that the temperature is between 15 and 60 ° C, preferably between 20 and 35 ° C, the incubation time is between 5 minutes and 24 hours, preferably between 10 minutes and 3 hours, and the pH is between 6.5 and 7.5.

3. Method according to claims 1 and 2, characterized in that the dispersion is a dispersion of organic particles, said particles being constituted by at least one organic polymer chosen from at least one homopolymer or a copolymer or their mixtures, resulting from the polymerization at least one monomer chosen from acrylamide and acrylate monomers, in particular N-alkylacrylamide and NN-dialkylacrylamide, such as N-isopropylacrylamide, N-methylacrylamide, N-ethylmethacrylamide, Nn-propylacrylamide , Nn-propylmethacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N, N-diethylacrylamide, N-methyl-N-isopropylacrylamide, N-methyl-Nn-propylacrylamide; alkyl acrylates and methacrylates in which the alkyl group comprises from 3 to 20 carbon atoms; styrene, methylstyrene, ethylstyrene, tert-butyl-styrene, chloromethylstyrene, vinyltoluene; their derivatives and the copolymers of these monomers with one another.

4. Method according to claims 1 and 2, characterized in that the dispersion is a dispersion of organic and inorganic particles, said organic particles being constituted by at least one organic polymer chosen from at least one homopolymer or a copolymer or their mixtures, derived of the polymerization of at least one monomer chosen from acrylamide and acrylate monomers, in particular N-alkylacrylamide and NN-dialkylacrylamide, such as N-isopropylacrylamide, N-methylacrylamide, N-ethylmethacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N, N-diethylacrylamide, N-methyl-N-isopropylacrylamide, N-methyl-Nn-propylacrylamide; alkyl acrylates and methacrylates in which the alkyl group comprises from 3 to 20 carbon atoms; styrene, methylstyrene, ethylstyrene, tert-butylstyrene, chloromethylstyrene; their derivatives and the copolymers of these monomers with one another; and said inorganic particles being chosen from particles of metal oxides, iron, titanium, cobalt, zinc, copper, manganese, nickel; magnetite; hematite, ferrites, such as manganese, nickel, manganese-zinc ferrites; cobalt, nickel alloys; zeolites; talc; clays such as bentonite and kaolin; alumina; silica; graphite; carbon black or other inorganic materials.

5. Method according to claims 1, 2, 3 and 4, characterized in that the envelope polymer is chosen from at least one homopolymer or a hydrophilic copolymer chosen from homopolymers or copolymers:

- resulting from the polymerization of at least one monomer chosen from monomers derived from acrylamide or from methacrylamide; acrylic acid, methacrylic acid; acrylate and methacrylate derivatives; allylamine; styrenic derivatives; on the condition, if it is a homopolymer, that it contains ionizable functional groups; in particular the copolymers or homoplymers of maleic anhydride and the homopolymers or copolymers of acryloxysuccinimide,

- polysaccharides, such as chitosan and poly galacturonic acid,

- polypeptides, such as polylysine and polyarginine, - linear or branched polyethyleneimine, and

- the dendrimers.

6. Method according to claim 5, characterized in that the envelope polymer is poly (maleic anhydride methyl vinyl ether), poly (N-vinylmorpholine-N-acryloxysuccinimide) or poly (N-vinylpyrrolidone- N-acryloxysuccinimide ).

7. Particulate reagent, having an affinity for biological compounds, comprising or consisting of organic and / or inorganic colloidal articulations, stable and functionalized, comprising a core and an envelope, characterized in that:

the core is essentially solid, organic and / or inorganic, and has functional groups X chosen from amine, hydroxyl, thiol, aldehyde, ester, anhydride, acid chloride, carbonate, carbamate, isocyanate and isothiocyanate groups or mixtures thereof , at least a fraction of which reacted with other functional groups of the envelope, and

the envelope consists of an ionizable polymer carrying functional groups Z and Z ′, which may be identical or different, chosen from the amine, carboxylic acid, ester, anhydride, aldehyde, thiol, disulfide, α-halocarbonyl, sulfonic acid groups, isocyanate, and isothiocyanate, which partially reacted with the functional groups X of the heart.

8. Reagent according to claim 7, characterized in that the core is organic and comprises at least one organic polymer chosen from at least one homopolymer or a copolymer or their mixtures, resulting from the polymerization of at least one monomer chosen from the monomers acrylamide and acrylate, in particular N-alkylacrylamide and N, N-dialkylacrylamide, such as N-isopropylacrylamide, N-methylacrylamide, N-ethylmethacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N -isopropylmethacrylamide, N-cyclopropylacrylamide, N, N-diethylacrylamide, N-methyl-N-isopropylacrylamide, N-methyl-Nn-propylacrylamide; alkyl acrylates and methacrylates in which the alkyl group comprises from 3 to 20 carbon atoms; styrene, methylstyrene, ethylstyrene, tert-butyl-styrene, chloromethylstyrene vinyltoluene; their derivatives and the copolymers of these monomers ente them and / or with other comonomers.

9. Reagent according to claim 7, characterized in that the core is organic and inorganic and comprises at least one organic polymer chosen from at least one homopolymer or a copolymer or their mixtures, resulting from the polymerization of at least one monomer chosen from acrylamide and acrylate monomers, in particular N-alkylacrylamide and N, N-dialkylacrylamide, such as N-isopropylacrylamide, N-methylacrylarnide, N-ethylmethacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N, N-diethylacrylamide, N-methyl-N-isopropylacrylamide, N-methyl-Nn-propylacrylamide; alkyl acrylates and methacrylates in which the alkyl group comprises from 3 to 20 carbon atoms; styrene, methylstyrene, ethylstyrene, tert-butylstyrene, chloromethylstyrene vinyltoluene; their derivatives and the copolymers of these monomers ente them and / or with other comonomers; and inorganic particles chosen from particles of metal oxides, iron, titanium, cobalt, zinc, copper, manganese, nickel; magnetite; hematite, ferrites, such as manganese, nickel, manganese-zinc ferrites; cobalt, nickel alloys; zeolites; talc; clays such as bentonite and kaolin; alumina; silica; graphite; carbon black or other inorganic materials.

10. Reagent according to Claims 7, 8 and 9, characterized in that the envelope polymer is chosen from at least one hydrophilic homopolymer or copolymer chosen from homopolymers or copolymers:

- resulting from the polymerization of at least one monomer chosen from monomers derived from acrylamide or from methacrylamide; acrylic acid, methacrylic acid; acrylate and methacrylate derivatives; allylamine; styrenic derivatives; on the condition if it is a homopolymer that it contains ionizable functional groups; in particular the copolymers or homoplymers of maleic anhydride and the homopolymers or copolymers of acryloxysuccinimide,

- polysaccharides, such as chitosan and poly galacturonic acid,

- polypeptides, such as polylysine and polyarginine,

- linear or branched polyethyleneimine, and - dendrimers.

11. Reagent according to claim 10, characterized in that the envelope polymer is poly (vinyl ether maleic anhydride), poly (N-vinylmorpholine-N-acryloxysuccinimide) or poly (N-vinylpyrrolidone-N- acryloxysuccinimide) .