CS241262B1 - Polymeric microparticles based on hydrophilic monomers and a process for their preparation - Google Patents

Abstract

The solution relates to polymeric microparticles based on hydrophilic monomers consisting of 25 to 99.5 wt. % hydrophilic monomers, selected from the group consisting of 2-hydroxyethyl methacrylate. 2-hydroxyethyl acrylate as well as the corresponding monoethers of *-dihydroxyoligo(oxyethylene) and methacrylic acid or acrylic acid, 2,5-dihydroxypropyl methacrylate, N-monosubstituted acrylamide, where the substituent may be alkyl with 1 to 5 carbon atoms or hydroxyalkyl with 1 to 6 carbon atoms, containing 1 to 2 OH groups, 4-vinylpyridine, 2-vinylpyridine, alkylamide and methacrylamide, 0.1 to 40 wt. ionogenic comonomers selected from the group consisting of methacrylic acid, acrylic acid, itaconic acid, maleic anhydride, 2-(diethylamino)ethyl methacrylate, 2-(dimethylamino)ethyl methacrylate, 2-(dimethylamino)ethyl acrylate, 2-(dimethylamino)ethyl acrylate, 2-(diethylamino)ethyl acrylate, p-diethylaminostyxene and 2-methacryloyloxyethylethylmethylammonium chloride, 0.1 to 45 wt.%. a crosslinker selected from the group consisting of N,?'-methnylenebis(acrylamide), N,'-ethylenebis3(acrylamide), 1,2.5-(triacryloyloxymethyl)propane, 1,5,5-triacryloylhydrotriazine, ethylenedimethacrylate, ethylenediacrylate, 2,2-oxydiethyl-dimethacrylate, dimethacrylates <,* -dihydroxyderivatives of oligoethylenes and 0.05 to 25 /5 wt. covalently bound hapten selected from the group consisting of N?methacryloylglycylglycyl-N-dinitrophenyllysine, N-methacryloylglycylphenylalanyl-N-dinitrophenyllysine.and Nmethacryloylglycylleucil-N-dinitrophenyllysine and optionally 0.05 to 7.5 wt. polymerizable fluorescent label selected from the group consisting of dansylally lamin, N-fluoresoein-H-allylthiourea and N-tetramethylrhodamine-N-allylthiourea

Description

The invention relates to polymeric microparticles based on hydrophilic monomers containing specific moieties facilitating their detection and allowing the binding of antibodies, as well as the use of these microparticles, in particular in immunology and parasitology and a process for their production.

The prior art synthetic microparticles have been produced by polymerization in the presence of initiators or emulsifiers, which cannot be completely removed from the resulting microparticle suspension. Also, the processes used so far have not made it possible to obtain a homogeneous particle size by polymerization. Also, the production of microparticles containing already polymerized haptens of precisely defined structures has not been described.

Said disadvantages of previously known and used in the microparticulate ^Z ZZ vv manufacturing process removes the present invention.

The present invention provides polymeric microparticles based on hydrophilic monomers containing specific moieties facilitating their detection and allowing the binding of antibodies, characterized in that they consist of 25 to 99.5% by weight. hydrophilic monomers selected from the group consisting of 2-hydroxyethyl methacrylate, 2-hydroxyethyl acrylate and the corresponding monoesters *, ^| v-dihydroxyoligo (oxyethylene) and methacrylic acid or acrylic acid; 2,3-dihydroxypropyl methacrylate, N-monosubstituted methacrylamide, N-monosubstituted acrylamide, Ν, Ν-disubstituted acrylamide, wherein the substituent may be C 1 -C 5 alkyl or C 1 -C 6 hydroxyalkyl containing 1 to 2 OH groups, % Of 4-vinylpyridine, 2-vinylpyridine, acrylamide and methacrylamide, 0.1 to 40 wt. ionogenic comonomers selected from the group consisting of methacrylic acid, acrylic acid, itaconic acid, maleic anhydride, 2- (diethylamino) ethyl methacrylate, 2- (dimethylamino) ethyl acrylate,

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% Of 2- (dimethylamino) ethyl acrylate, 2- (diethylamino) ethyl acrylate, p-diethylaminostyrene and 2-methacryloyloxyethyltrimethylammonium chloride, 0.1 to 45 wt. a crosslinking agent selected from the group consisting of N, N-methylenebis (acrylamide), N, N-ethylenebis (acrylamide), 1,2,3- (triacryloyloxymethyl) propane, 1,3,5-triacryloylhexahydrotriazine, ethylene dimethacrylate, ethylenediacrylate, 2,2 % oxydiethyl dimethacrylate, dimethacrylates, oligoether dihydroxy derivatives and 0.05 to 25 wt. by a covalent bond of the bound hapten selected from the group consisting of N-methacryloylglycylglycyl-N-dinitrophenyllysine and N-methacryloylglycylphenylalanyl-N-dinitrophenyllysine and N-methacryloylglycylleucyl-N-dinitrophenylysine, and optionally Q-5, and optionally Q-5 to 7-methacryloyl. polymerizable fluorescent labels selected from the group consisting of dansylalylamine, N-fluorescein-N-allylthiourea and N-tetramethylrhodamine-. N-allyithioureas.

The process for preparing the microparticles according to the invention consists in copolymerizing a solution of 1 to 10% by weight of the radicals formed by irradiation. % monomer mixture and 90 to 99 wt. % of water or a mixture thereof with 0 to 20% by volume of C1-C4 alcohols or other water-miscible solvent, wherein the monomer mixture is composed of 25 to 99.5% by weight of water; % hydrophilic monomer (s), 0.1 to 40 wt. % ionogenic comonomers, 0.1 to 45 wt. % of a crosslinker, 0.05 to 25 $ wt. % of polymerixable hapten, and optionally 0.05 to 75% by weight. polymerizable fluorescent labels, wherein the individual monomer components are as defined above.

The monomer solution may additionally contain up to 2% by weight. Poly (N-monosubstituted acrylamide), a poly (N-monosubstituted acrylamide), wherein the substituent can be a C1-C4 alkyl or a 1-5C hydroxyalkyl, can also be used. carbon atoms containing 1 to 2 OH groups, poly (vinyl) alcohol), polyacrylamide, polyesters (ethylene) and polyesters (oxypropylene), as well as monoalkyl ethers. The molecular weight of the additives is preferably in the range of 5,000 to 250,000 daltons. The sterile additive makes it possible to influence the particle size. It also makes it possible to carry out co-polymerization at higher concentrations. It can be evaluated as a particle stabilizer.

As the water-miscible solvent, in addition to the C1-C4 alcohols, 1,4-dioxane, acetone, dimethyl sulfoxide, dimethylformamide, ethyl241, 262 can be used.

-3.acetate etc.

AND

Hydrophilic monomers which may be used are 2-hydroxyethyl methacrylate, 2-hydroxyethyl acrylate, as well as the corresponding monoesters of α, β-dihydroxyoligo (oxyethylene) and methacrylic acid or acrylic acid; 2,3-dihydroxypropyl methacrylate, N-monosubstituted methacrylamide, N-monosubstituted acrylamide, N, N-disubstituted acrylamide, wherein the substituent may be C 1 -C 5 alkyl or C 1 -C 6 hydroxyalkyl containing 1 to 2 OH 4-vinylpyridine, 12-vinylpyridine; acrylamide and methacrylamide. C-labeled monomers may also be used.

Ionogenic comonomers are methacrylic acid, acrylic acid, itaconic acid, maleic anhydride, 2- (diethylamino) ethyl methacrylate, 2- (dimethylamino) ethyl methacrylate, 2- (dimethylamino) ethyl acrylate, 2- (diethylamino) ethyl- acrylate, p-diethylaminostyrene, 2-methacryloyloxyethyltrimethylammonium chloride.

N, N-methylenebis (acrylamide), N, N-ethylenebis (acrylamide),], 2,3- (triacryloyloxymethyl) propane, 1,3,5-triacryloylhexahydrotriazine, ethylenedimethacrylate, ethylenediacrylate, 2,2- (triacryloyloxymethyl) propane are preferred as crosslinkers. oxydiethyldimethacrylate as well as dimethacrylates of the oligoether dihydroxy derivatives. The amount of crosslinker used is controlled by the desired degree of crosslinking. It is dependent on the ability of the crosslinker 1a to form cross-links.

As polymerizable fluorescent agents, for example, dansyl] glylylamine, N-fluorescein-N-allylthiourea, N-tetramethylrhodamine-N-allylthiourea and N-acridine-N-allylthiourea.

As polymerizable haptens, for example, N-methacryloylglycylglycyl-N'-dinitrophenyllysine, N-methacryloylglycylglycylarsanilic acid, N-methacryloylglycylphenylalanyl-N'-dinitro-N-methacryloylin N-methacryloylsilyl and N-methacryloylsin.

In the polymerization, the monomer mixture (polymerization mixture) is filled into a polymerization vessel, for example a glass vial, purged with nitrogen or argon for about 15-20 minutes and sealed or otherwise sealed. At a temperature of preferably 0 ° C to 40 ° C, the vial is placed in the center of the cobalt source and irradiated so that the dose is 0.15Mrad.

After irradiation, the ampoule is opened, the contents of 10x are decanted with an excess of distilled water (or a mixture of water and a water-miscible solvent and then distilled water) and stored at 4 ° C.

There are several methods for binding antibodies. For example, the interaction of antibodies with the haptenic groups present in the structure, the microparticles (see Example 1) may be utilized, or the functional groups present in ionogenic comonomers - for example -COOH groups in the methacrylic acid molecule - may be utilized. To these groups, the bioactive molecules can be coupled with N-cyclohexyl-N, 2- (4-morpholinyl) ethylcarbodiimide-methyl p-toluenesulfonates, Woodward's reagent, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline and the like in the peptide chemistry of used condensing agents.

The microparticles prepared according to the invention are very suitable for use in detecting specific membrane traits and for phagocytic assays, particularly of cells involved in immune responses, but also of other cell types. Unlike many of the techniques used hitherto, both particle binding to the receptor of interest and phagocytosis mediated by the receptor can be detected simultaneously. Particularly advantageous seems to be the use of particles with a polymerized fluorescent label, which, when detected in a fluorescent microscope, distinguish phagocytosed from non-phagocytosed particles by using a suitable quencher of the non-phagocytosed particles. The differentiation of phagocytosed from non-phagocytosed microparticles is performed after the fluorescence quenching of the fluorescent modification of the microparticles by a quencher, for example crystal violet.

The advantage of incorporating hapten of a precisely defined structure by the process of the invention is that haptens are not released from the microparticles into the environment and retain their immunogenicity and functionality. Binding of bioactive molecules by direct polymerization, but also by means of condensing agents, is very simple and stable. The durability of the suspension, alone and with the attached haptens, was verified at the Immunology Department of the Institute of Chemical Technology of the Czech Academy of Sciences from April 1981 to February 1984.

The following non-limiting examples illustrate the invention.

Example 1

Reaction of 5 tetramethylrhodamine isothiocyanate with an excess of allylamine in acetone at 50 ° C prepared N-tetramethylrhodamine-N * -allylthiourea. Acetone and excess allylamine were distilled off under reduced pressure. To the oil obtained was added 0.4 ml of a monomer mixture (76.9 parts by weight of 2-hydroxyethyl methacrylate, 0.1 parts by weight of ethylenedimethacrylate, 2 parts by weight of methylene-bis-acrylamide, 20 parts by weight of methacrylic acid and 1% by weight of methacrylic acid). (N-methacryloylglycylglycyl-N, N-dinitrophenyllysine) and 19.6 ml of distilled water. The obtained homogeneous solution was filtered, filled into an ampoule, bubbled through for 15 min. nitrogen and irradiated in a ° ° C source (1,2 Mrad). The resulting suspension was poured into excess distilled water, decanted. A suspension of 2 µm particles was obtained.

These particles were reacted with the maximum subagglutination dose of murine monoclonal anti-trinitrophenyl antibodies, the IgE isotype. The particles were washed (600g, 10 min), resuspended and stored at 4 ° C. They have been used to detect cell surface immunoglobulin receptors and phagocytic assays.

Example 2 10 g of a mixture consisting of 77.8% by weight. 2-hydroxyethyl methacrylate, 2% by weight of methylene-bis-acrylamide, 0.2% by weight of polyethylene oxide monomethyl ether of = 5,000 and 20% by weight of methacrylic acid were dissolved in 190 ml of distilled water. The polymerization was carried out as in Example 1. 1.2 µm particles were obtained, 50 µg of microparticles (dry weight) were resuspended in 1 ml of purified C 3 complement component (1 mg / ml). After cooling to

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At 4 ° C, 5 mg of N-cyclohexyl-N ^from -? - 2- (4-morpholinyl) ethyl] carbodiimide methyl p-toluenesulfonate was added slowly with vigorous shaking. The pH was immediately adjusted to 7-10 and the microparticles were incubated for 10 hours at 4 ° C from shaking. After washing several times in saline (250 g, 15 minutes) and diluting to a concentration of 10 particles / ml, 0.1 ml of the diluted microparticles were mixed with 10 µl (in 0.1 ml) and incubated for 60 min. at room temperature. Cells with adhered 3 or more microparticles were considered to carry the receptor for the C 3 complement component.

Example 3 mg of dansyallylamine was dissolved in 4.5 ml of a mixture of monomers consisting of 50% by weight. % 2-hydroxyethyl methacrylate, 20 wt. N, N-diethyl acrylamide, 4 wt.% Triethylene glycol dimethacrylate and 26 wt.% Acrylic acid. To this solution was added 245 mL of distilled water. The polymerization was then carried out as in Example 1.

Example 4

Reaction of 3 mg fluorescein isothiocyanate (isomer i) with an excess of allylamine in acetone at 40 ° C prepared N-fluorescein-N * -allylthiourea. Acetone and excess allylamine were distilled off under reduced pressure. 0.6 ml of monomer mixture (77 parts by weight of 2-hydroxyethyl methacrylate, 2 parts by weight of methylene-bis-acrylamide, 20 parts by weight of methacrylic acid and 1 part by weight of N-methacryloylglycylglycyl-N, N-dinitrophenyllysine) were added to the obtained crystals. and 15.4 ml of distilled water. The polymerization proceeded as in Example 1. These particles were incubated with the maximum subagglutination dose of murine monoclonal anti-trinitrophenyl IgM isotype antibodies (22 ° C, 1 hr). After the particles were washed, resuspended and stored in a refrigerator. They were used to detect Fc receptors on the cell surface - after incubation of the cells with the modified microparticles (22 ° C), the cells were observed under a fluorescence microscope. Cells that had adhered 3 or more microparticles were considered to contain Fc receptors.

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Example 5

Two stock solutions were prepared. Solution A, consisting of 0.03 g of N, N-methylenebis (acrylamide), 1.309 g of 2-hydroxyethyl methacrylate and 0.308 g of 2- (diethylamino) ethyl methacrylate. Solution B, consisting of 1,589 g of polyethylene oxide methyl ether (m. V. 5,000), dissolved in 38,2 ml of water. 0.4 ml of solution A, 4 ml of solution B and 35 ml of water were introduced into a glass vial. The polymerization was then carried out as in Example 1.

Example 6 mg of dansylallylamine was dissolved in a monomer mixture consisting of 55 wt. % 2-liydroxyethyl 1-methacrylate, 15 wt. % N-ethyl acrylamide, 5 wt. 1,2,3- (triacryloyloxymethyl) propane and 25% by weight of 4-vinylpyridine. To this solution was added 230 mL of distilled water. The polymerization was then carried out as in Example 1.

Claims (6)

SUBJECT OF THE INVENTION CLAIMS 1. Polymeric microparticles based on hydrophilic monomers having a specific group, facilitating their detection and allowing antibodies, characterized in that they consist of 25 to 99% of hydrophilic monomers selected from the group consisting of 2-hydroxyethyl methacrylate, 2-hydroxyethyl acrylate as well as the corresponding monoesters *, α'-dihydroxyoligo (oxyethylene) and methacrylic acid or acrylic acid, 2,3-dihydroxypropyl methacrylic acid, N-monosubstituted methacrylamide, N-monosubstituted acrylamide, N, N-disubstituted % of acrylamide, wherein the substituent may be C 1 -C 5 alkyl or C 1 -C 6 hydroxyalkyl containing 3 to 2 OH groups, 4-vinylpyridine, 2-vinylpyridine, acrylamide and methacrylamide, 0.1 to 40 wt. ionogenic comonomers selected from the group consisting of methacrylic acid, acrylic acid, itaconic acid, maleic anhydride, 2- (diethylamino) ethyl methacrylate, 2- (dimethylamino) ethyl acrylate, 2- (diethylamino) ethyl acrylate, p-diethylaminostyrene, and 2-methacryloyloxyethyl trimethyl ammonium. % chloride, 0.1 to 45 wt. a crosslinker selected from the group consisting of Ν, Ν-methylenebis (acrylamide), N, N-ethylenebis (acrylamide), 1,2,3-tri (acryloyloxymethyl) propane, 1,3,5-triacryloylhexahydrotriazine, ethylenedimethacrylate, ethylenediacrylate, % Of 2,2-oxydiethyl dimethacrylate, dimethacrylate *, oligoether t-dihydroxy derivative and 0.05 to 25 wt. covalently-linked hapten selected from the group consisting of N-N? methakryloylglycylglycyl -dinitrofenyllysinu methakryloylglycylfenylalanyl and N-N ^t and N -dinitrofenyl1ysinu methakryloylglycylleucyl N? -dinitrofenyllysinu and optionally 0.05 to 7 »5% by weight. polymerizable fluorescent labels selected from the group consisting of dansylallylamine, N-fluorescein-N-allylthiourea and N-tetramethylrhodamine-N-allylthiourea. 2. The polymer microparticle of claim 2 wherein the hydrophilic monomer is 2-hydroxyethyl methacrylate. -1241 282 3. The polymeric microparticle of claim 1 wherein the ionogenic comonomer is methacrylic acid. 4. The polymeric microparticle of claim 1 wherein the crosslinker is N, N-methylbenzenes (acrylamide). 5. A process for producing microparticles based on hydrophilic monomers comprising specific moieties facilitating their detection by allowing the deposition of an irradiation / antibody-binding radical according to claim 1, wherein the solution comprises from 1 to 10% by weight of the composition. mixtures of monomers and 9θ to 99% by weight or mixtures thereof with 0 to 20 # vol. . % of alcohols or jintite with a water-miscible solvent, wherein the monomer mixture is comprised of 25 to 99.5% by weight. % hydrophilic monomer (s), 0.1 to 40 wt. % ionogenic comonomers, 0.1 to 45 wt. crosslinker, 0.05 # to 25 # wt. % of polymerizable hapten and optionally 0.05 to 7 > polymerizable fluorescent labels, wherein the individual monomer components are as defined in point 1. 6. A process according to claim 5, wherein the monomer solution additionally contains up to 2% by weight. a polymeric additive, preferably [- N- (2-hydroxypropyl) methacrylamide].