WO2002092652A1

WO2002092652A1 - Poly(n-isopropylacrylamide) latex comprising nitrile functions on the surface thereof

Info

Publication number: WO2002092652A1
Application number: PCT/FR2002/001570
Authority: WO
Inventors: Thierry Delair; Guangchang Zhou; Abdelhamid Elaissari
Original assignee: Bio Merieux
Priority date: 2001-05-14
Filing date: 2002-05-07
Publication date: 2002-11-21
Also published as: FR2824562A1

Abstract

The invention relates to a method of preparing hydrophilic and heat-sensitive polymer particles carrying polar functional groups by means of radical co-polymerisation. The inventive method is characterised in that it involves the homopolymerisation of a water-soluble monomer functionalised by said polar groups in order to obtain a hydrophobic homopolymer functionalised by said groups and the subsequent co-polymerisation with the non-functionalised hydrophilic and heat-sensitive polymer. The invention also relates to the particles thus obtained.

Description

POLY LATEX (N-ISOPROPYLACRYLAMIDE) HAVING NITRILE FUNCTIONS ON THE SURFACE

The present invention relates to hydrophilic, thermosensitive latexes based on poly (N-isopropylacrylamide), functionalized allowing the production of particulate supports.

These functionalized latexes have many applications due to the thermosensitivity of poly (N-isopropylacrylamide), in particular in the field of diagnostics, for separation by chemical immobilization or adsorption of biological molecules then assay.

Biological molecules can be immobilized by covalent bond or physical adsorption on the surface of the particles. Poly (N-isopropylacrylamide) particles comprising a core and a surface carrying functional groups, such as the carboxylic or amino acid functions, have been developed as solid supports for the immobilization of biological molecules.

Particles, comprising a magnetic charge, comprising a core based on a polymer, an inner layer based on a second heat-sensitive polymer in which a magnetic material is distributed, and an outer layer, said encapsulation layer optionally functionalized with based on a polymer capable of interacting with at least one biological molecule, are described for example in FR-A-2749082. The polymer of the encapsulation layer capable of interacting with a biological molecule is preferably a hydrophilic polymer optionally functionalized by one or more groups chosen from the carboxylic, aldehyde, thiol and amino functions.

These particles are used as solid support for the immobilization of biological molecules such as enzymes and proteins, in particular in the form of latex. Different modes of polymerization can be used to prepare these supports, by polymerization we mean both copolymerization and homopolymerization. For example, polymerization in a closed reactor known as “batch” polymerization is known, in which the monomers are introduced into the reactor before the start of the polymerization reaction with the other ingredients and without subsequent addition. This method is effective for the copolymerization of a mixture of hydrophobic and hydrophilic monomers because the hydrophobic monomer mainly forms the core and the hydrophilic monomer forms the shell if the polymerization takes place in the aqueous phase.

Polymerization in "shot" is known, this technique can be used when we know the kinetics of polymerization in a closed reactor. When the polymerization in a closed reactor is in progress, an additional quantity of functional monomer alone or in the presence of a mixture of monomers is introduced into the reactor in a controlled manner, making it possible to promote the incorporation of the functional monomer on the surface of the particles. The selection of the experimental conditions (degree of conversion at the time of addition, composition and concentration of the mixture of monomers) makes it possible to optimize the surface yields.

Also known is seed polymerization which consists in introducing the functional monomer or a mixture of monomers into a reactor containing an already constituted and characterized latex, the functional monomer or the mixture of monomers can be added to the seed in a single step or semi-continuous.

However, for the dosages to be precise, it is necessary that the immobilized biomolecules can retain their specific biological activity, and this biological activity, dependent on steric factors and of orientation, can possibly be reduced, or even eliminated, by immobilization techniques.

An attempt to improve the immobilization technique was carried out by creating a technique using regioselective interactions, such as for example dipolar interactions. These dipolar interactions using specific regions of the biomolecules carrying polar groups, the predictability of the conservation of the biological activity can be good.

The polymers carrying nitrile functions have been found to be particularly interesting in this new approach, see G. ZHOU et al., Colloid Polym. Sci., 276: 1131-1139 (1998). The particles maintain lower critical solubility temperatures (LCST) of the same order of magnitude as those of nonfunctionalized latexes, and can be prepared by the methods conventionally used in radical polymerization such as the methods of preparation by precipitation, dispersion, suspension and emulsion.

However, only the polymerization process by the “shot” polymerization technique makes it possible to obtain poly (N-isopropylacrylamide) latexes functionalized by nitrile functions comprising particles on which the nitrile functions are sufficiently numerous at the surface.

As previously specified, this “shot” polymerization technique can only be used when the polymerization kinetics are known.

The polymerization process in a closed reactor known as “batch” polymerization applied to the preparation of poly (N-isopropylacrylamide) latex functionalized with nitrile functions does not make it possible to obtain particles on which the nitrile functions are sufficiently numerous at the surface and does not provide sufficient returns. In fact, a large part of the functional monomer risks being lost either inside or in the form of a water-soluble polymer. It is also noted that due to the high solubility in water of monomers of a polar nature, such as the acrylonitrile and methacrylonitrile derivatives, the copolymerization will lead to smaller particles, with a limited conversion rate. In order for these latexes to be able to be used for immobilization by dipolar interaction and for immobilization to be effective, the respective polar functional groups of the antibodies and of the particles must be able to be in contact with each other easily.

The polar functional groups of the particles must therefore be present on the surface of the particles, and in large numbers, so that the statistically dipole interactions can take place and that the immobilizations are effective.

To achieve this objective, with reaction times compatible with industrial production and sufficient yields, a process of Polymerization comprising a step of prepolymerization of the monomer functionalized by polar groups has been developed.

This prepolymerization step makes it possible to synthesize homopolymers comprising polar functions but being insoluble in water.

It thus makes it possible to create functionalized radical homopolymer chains, that is to say comprising polar functions, but which can then be polymerized by techniques such as “batch” polymerization. This prepolymerization step is applicable to all the monomers functionalized by polar functional groups, soluble in water, the homopolymers of which are insoluble in water.

It is possible, for example, to prepolymerize water-soluble functionalized monomers, that is to say comprising a polar function, such as the acrylonitrile and methacrylonitrile derivatives, in order to obtain homopolymers insoluble in water but comprising a polar function and capable of subsequently reacting on a non-functionalized polymer. .

The method according to the invention makes it possible to obtain particles comprising nitrile functions in sufficient number at the surface and with correct yields.

An object of the invention is a process for the preparation of thermosensitive and hydrophilic polymer particles carrying polar functional groups by radical copolymerization, characterized in that a water-soluble monomer functionalized with said polar groups is homopolymerized to obtain a hydrophobic homopolymer functionalized with said polar groups and then copolymerized with the non-functionalized thermosensitive and hydrophilic polymer.

The process for the preparation of thermosensitive and hydrophilic polymer particles carrying polar functional groups by radical polymerization according to the invention can be defined as a process by which a non-functionalized thermosensitive and hydrophilic polymer is copolymerized with a hydrophobic homopolymer functionalized by said polar functional groups .

The method of the invention further comprises the following characteristics considered alone or as a mixture. According to the invention, the polar functional groups are nitrile functions and the functionalized water-soluble monomer is chosen from acrylonitrile and methacrylonitrile derivatives, for example acrylonitrile.

According to one embodiment, the functionalized water-soluble monomer is present in amounts of between 15 and 30% relative to the total amount of the other monomers.

According to the invention the non-functionalized thermosensitive and hydrophilic polymer is obtained by crosslinking of a monomer chosen from N-alkylacrylamides and N, N-dialkylacrylamides, and preferably from N-isopropylacrylamide, N-ethylmethacrylamide, Nn-propylacrylamide , Nn-propylmethacrylamide, N-isopropylmethacrylamide, N- cyclopropylacrylamide, N, N-diethylacrylamide, N-methyl-N- isopropylacrylamide, N-methyl-Nn-propylacrylamide, and, for example poly (N -isopropylacrylamide). The crosslinking agent is water-soluble and is chosen from N, N-methylene bisacrylamide (MBA), ethylene glycol dimethacrylate.

The non-functionalized polymer is obtained by reaction of the non-functionalized monomer with the crosslinking agent, for example Poly (N-isopropylacrylamide) obtained by the action of MBA on NIPAM in the presence of an initiator.

The functionalized monomer is chosen from acrylonitrile and methacrylonitrile derivatives, the functionalized monomer preferably being acrylonitrile.

Another object of the invention relates to the particles of thermosensitive and hydrophilic polymer carrying polar functional groups capable of being obtained by a process of preparation by radical copolymerization characterized in that a water-soluble monomer functionalized by said polar groups is homopolymerized obtaining a hydrophobic homopolymer functionalized by said polar groups and then copolymerizing with the non-functionalized thermosensitive and hydrophilic polymer.

The invention also relates to a method for isolating from a liquid sample at least one biological molecule, according to which:

- particles according to the invention are available, - said sample is brought into contact with said particles

- by incubation, the particles of the sample are separated, and a method of selective immobilization of specific biological molecules from a sample according to which:

- particles are available according to the invention, - said sample is brought into contact with said particles and

- the selectivity and the specificity are controlled by temperature control, the temperature being a critical parameter of the interaction between biological molecule and particle.

Desorption is promoted by lowering the temperature to a temperature below the LCST.

It also relates to a reagent for the isolation of biological molecules comprising a dispersion in an aqueous medium of particles according to the invention.

The expression “isolate a biological molecule” according to the invention includes the separation, detection, purification of a biological molecule, the enrichment of a fraction in a biological molecule, according to a specific or non-specific isolation method specific, qualitatively and / or quantitatively, directly or indirectly, for example by means of a ligand attached to the particles. The term “biological molecule” means a compound chosen in particular from proteins, antibodies, antibody fragments, antigens, polypeptides, enzymes, haptens, nucleic acids and their fragments, and which has at least one recognition site. allowing it to react with a target molecule of biological interest. The term "nucleic acid" means a sequence of at least

2 deoxyribonucleotides or ribonucleotides optionally comprising at least one modified nucleotide, for example at least one nucleotide comprising a modified base such as inosine, methyl-5-deoxycytidine, dimethylamino-5-deoxyuridine, deoxyuridine, diamino-2, 6-purine, bromo-5-deoxyuridine or any other modified base allowing hybridization. This polynucleotide can also be modified at the level of the intemucleotide bond. The nucleic acid can be natural or synthetic; an oligonucleotide, a polynucleotide a nucleic acid fragment, a ribosomal RNA, a messenger RNA, a transfer RNA, a nucleic acid obtained by an enzymatic amplification technique such as PCR (Polymerase Chain Reaction), LCR (Ligase Chain Reaction), RCR (Repair Chain Reaction), 3SR (Self Sustained Sequence Replication), NASBA (Nucleic Acid Sequence-Based Amplification) and TMA (Transcription Mediated Amplification). The nucleic acids generated by an enzymatic amplification technique are referred to as amplicons. By “polypeptide” is meant a chain of at least two amino acids. By amino acids is meant the primary amino acids which code for proteins, amino acids derived after enzymatic action such as trans-4-hydroxyproline and natural amino acids but not present in proteins such as norvaline, N-methyl- Leucine, Stalin (see Hunt S. in Chemistry and Biochemistry of the amino acids, Barett GC, ed., Chapman and Hall, London, 1985), amino acids protected by chemical functions usable in synthesis on solid support or in liquid phase and unnatural amino acids.

The term "hapten" designates non-immunogenic compounds, that is to say incapable by themselves of promoting an immune reaction by production of antibodies, but capable of being recognized by antibodies obtained by immunization of animals in known conditions, in particular by immunization with a hapten-protein conjugate. These compounds generally have a molecular mass of less than 3000 Da, and most often less than 2000 Da and can be, for example, glycosylated peptides, metabolites, vitamins, hormones, prostaglandins, toxins or various drugs, nucleosides and nucleotides.

The term "antibody" includes polyclonal or monoclonal antibodies, antibodies obtained by genetic recombination, and antibody fragments such as Fab or F (ab ') 2-

The term "antigen" denotes a compound capable of generating antibodies.

The term "protein" includes holoproteins and heteroproteins such as nucleoproteins, lipoproteins, phosphoproteins, metalloproteins and both fibrous and globular glycoproteins in their characteristic conformational form. Example 1: Preparation of seeded Polv (N-isopropylacrylamide) latex

A mixture of N-isopropylacrylamide (NIPAM) and N'N-methylene bisacrylamide (MBA) is dissolved in deionized water, free of oxygen in a 500 ml reactor equipped with mechanical stirring, a refrigerant , a thermocouple and under a nitrogen atmosphere.

To completely remove the oxygen from the monomer phase, the solution is stirred at 220 rpm under a stream of nitrogen. The polymerization is initiated by the addition of 20 ml of an aqueous solution of potassium persulfate (KPS) at 70 ° C., under a stirring speed of 303 rpm, the polymerization is carried out for two hours.

The poly (N-isopropylacrylamide) latex (PNIPAM) is then purified by successive centrifugations and redispersions.

An example of the quantities of reagents brought into contact is given in Table 1.

Table 1

Example 2 Preparation of Polv (N-isopropylacrylamide) latex comprising nitrile functional groups on the surface

The seeded Poly (N-isopropylacrylamide) latex is deoxygenated by purging with nitrogen for 30 minutes at 40 ° C.

Deionized, oxygen-free water is placed in a 100 ml reactor equipped as in Example 1, also deoxygenated by nitrogen purging for 20 minutes, then an aqueous acrylonitrile solution is added to the reactor in which the addition of nitrogen is stopped.

When the temperature reaches 60 ° C. an aqueous solution of potassium persulfate (KPS), the initiator, is added. The prepolymerization of acrylonitrile (AN) is continued for 5 minutes with stirring at 220 rpm, then the dispersion of Poly (N-isopropylacrylamide) (PNIPAM) prepared in Example 1, and free of oxygen, is added in the closed reactor.

The polymerization reaction is then continued for two hours.

The functionalized latex obtained is then purified according to the method described in the previous procedure.

The amounts of reagents involved in this preparation are given in Table 2 below. Two different batches were prepared. They are coded ZG-1 and ZG-2.

Table 2

* Solid content of PNIPAM latex: 6.37%

Example 3 Influence of the Quantity of Acrylonitrile Used on the Size of the Particles

Particle size is measured using diameter measurement by dynamic light scattering.

The results obtained are collated in Table 3 below.

It is observed that the sizes of the latex particles carrying the code ZG-1 at both 20 ° C and 45 ° C are larger than the sizes of the latex particles carrying the code ZG-0.

These data make it possible to conclude that the nitrile functions have been introduced predominantly on the surface of the polymer particles. Table 3

DIAMETER OF PNIPAM PARTICLES CARRYING NITRILE FUNCTIONS

SURFACE

"Poly (N-isopropylacrylamide) seeded latex

EXAMPLE 4 Immobilization of an Antibody on the Surface of Functionalized Particles of Polv (N-isopropylacrylamide) by Nitrile Functions

All adsorption tests were performed in 2 ml Eppendorf tubes.

To determine the amount of immobilized antibodies (Ab) on the latex particles, the conventional method of depletion is used.

A given volume of latex particles is added to the tube with a given volume of antibody solution and then made up with a buffer solution (PBS) to 500 ml of final volume.

The mixture is then stirred at a temperature of 32 ° C for three hours, then centrifuged at 14,000 rpm at 20 ° C for 20 minutes to separate the latex particles.

In order to determine the residual antibody concentration in the supernatant, it is extracted and analyzed using a UVIKON spectrophotometer. The quantity of immobilized antibodies (Ac) is calculated by difference between the initial and residual concentration of antibodies in the supernatant according to the following equation Ns = V [(Ci-Cf) / (MS)] in which V is the volume of the solution in ml, Ci (mg-mf ¹ ) and Cf (mg-ml ¹ ) the initial and final concentrations of antibodies in the solution, M (g) the weight S (m ² g- ¹ ) the specific surfaces of latex particles. The results obtained enabling the impact of the quantity of nitrile functions contained in the functionalized PNIPAM latex particles to be quantified on the immobilization of antibodies are collated in Table 4 below.

Table 4

EFFECT OF THE QUANTITY OF NITRILE FUNCTIONS CONTAINED IN FUNCTIONALIZED LATEX PARTICLES ON ADSORPTION

ANTIBODIES AC *

Initial antibody concentration in the solution: 19.20 x 10- mg / ml, total volume 500 μl, interaction time: 3 hours, temperature: 32 ° C.

a) Calculated according to equation 1 using the specific surface of the two functionalized latexes (15.49 mg ^"1 and 10.18 m ² g ¹ for ZG2).

Claims

1. Process for the preparation of thermosensitive and hydrophilic polymer particles carrying polar functional groups by radical copolymerization, characterized in that a water-soluble monomer functionalized with said polar groups is homopolymerized to obtain a hydrophobic homopolymer functionalized with said groups and then copolymerized with the non-functionalized thermosensitive and hydrophilic polymer.

2. Process for the preparation of thermosensitive and hydrophilic polymer particles carrying polar functional groups by radical polymerization, characterized in that a non-functionalized thermosensitive and hydrophilic polymer is copolymerized with a hydrophobic homopolymer functionalized by said polar functional groups.

3. Preparation process according to any one of claims 1 to 2, characterized in that the polar functional groups are nitrile functions.

4. Method according to any one of claims 1 to 3 characterized in that the functionalized water-soluble monomer is chosen from acrylonitrile and methacrylonitrile derivatives.

5. Method according to any one of claims 1 to 4 characterized in that the functionalized water-soluble monomer is acrylonitrile.

6. Method according to any one of claims 1 to 5 characterized in that the functionalized water-soluble monomer is present in amounts of between 15 and 30% relative to the total amount of the monomers.

7. Method according to any one of claims 1 to 2 characterized in that the non-functionalized thermosensitive and hydrophilic polymer is obtained by crosslinking of a monomer chosen from N-alkylacrylamides and N, N-dialkylacrylamides, and preferably from N-isopropylacrylamide, N-ethylmethacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N, N- diethylacrylamide, N-methyl-N-isopropylacrylamide, N-methyl-Nyl-propyl

8. Method according to claim 7 characterized in that the non-functionalized thermosensitive and hydrophilic polymer is Poly (N- isopropylacrylamide).

9. Thermosensitive and hydrophilic particles carrying polar functional groups characterized in that they are capable of being obtained by a process according to any one of claims 1 to 8.

10. A method for isolating from a liquid sample at least one biological molecule, according to which:

- particles are available according to claim 9,

- said sample is brought into contact with said particles by incubation,

- The particles are separated from the sample.

1 1. Reagent for the isolation of biological molecules comprising a dispersion in an aqueous medium of particles characterized in that it consists of particles according to claim 9.

12. Method for selective immobilization of specific biological molecules from a sample according to which:

- particles are available according to claim 9,

- said sample is brought into contact with said particles and