DE19800294A1 - Inductively heatable polymer encapsulated magnetic particles for coupling bio-ligands - Google Patents

Abstract

Magnetic particles (I) comprises an inductively heatable magnetic core, completely encapsulated in a polymeric matrix on which ligands are chemically coupled. An Independent claim is included for the production of the magnetic particles, comprising encapsulating nanoparticulate or microparticulate magnetic particles in a polymer matrix by emulsion or suspension polymerization, precipitation methods (e.g. a solvent evaporation technique) or suspension crosslinking; and forming reactive groups coupled with bioligands on the surface.

Description

The invention relates to inductively heatable particles, which are characterized in that fine distributed magnetic particles are encapsulated in a functional polymeric matrix, which in the It is able to bind biomolecules, cells, bacteria, toxins, fungi or viruses.

Magnetic colloids, consisting predominantly of magnetite (Fe₃O₄), iron oxide (Fe₂O₃) or Iron oxyhydroxide (FeOOH) exist and have a particle size of 5-100 nm and u. a. as a contrast agent in NMR diagnostics, as information storage media, sealing or Damping agents are used are generally known from the following publications: PS 5,492,814; USPS 4,827,945; DE-OS 38 08 000, US-PS 3,215,572, DE-OS 39 33 210, US-PS 3,917,538, DE-OS 39 33 210, US-PS 4,329,241, PCT / EP 0 275 285, EP 0 586 052. As a rule, iron (III) and iron (II) salt solutions with varying molar Ratios (2: 1, 0.5: 1 to 4: 1) by adding bases or by applying heat in accordingly colloidal magnetic dispersions ("magnetic colloids") transferred. To be a special one to prevent agglomeration of the magnetic colloids caused by the Van der Waals forces, are added to these surfactants, commonly called "Surfactants", "emulsifiers", "stabilizers", "complexing agents", "surfactants" or "dispersants" are known and are hereinafter referred to as "stabilizers", which practically prevents the colloid from settling in aqueous dispersion. Such stabilized colloidal dispersions are also known under the name "ferrofluids" (cf. Kaiser and Miskolczy, J. Appl. Phys., 413, 1064. 1970). They are also offered commercially today (Ferrofluidics Corp., USA, Advanced Magnetics, USA, Taibo Co., Japan, Liquids Research Ltd, Wales, BASF, Germany, Schering AG, Germany). The stabilizers used are either cationic or anionic in nature or non-ionic. Suitable connections for this are e.g. For example: alkylaryl polyether sulfates, lauryl sulfonate, alkylaryl polyether sulfonates, Phosphate esters, alcohol ether sulfates, citrates, oleic acid, alkylnaphthalene sulfonates, Polystyrene sulfonic acid or petroleum sulfonates as anionic substances, Dodecyltrimethylammonium chloride as cationic surfactants and nonylphenoxypolyglycidol,  Kerosene, alkylaryloxypolyäthoxyäthanole, nonylphenol or polyethylene glycols as non-ionic Substances. Another class of substances that have proven themselves as colloid stabilizers, are: polysaccharides and synthetic polymers such as polyvinyl alcohol, polyoxyethylene or Proteins (see U.S. Patent 3,917,538, PCT / WO 89/11154, U.S. Patent 4,827,945).

The particle sizes shown with the help of the aforementioned preparation methods Experience has shown that magnetic colloids depend on the concentration of the base, the type of base, the Temperature as well as the salt ratio and the ion concentration.

A new process for the preparation of stable colloidal dispersions from magnetite and γ-Fe₂O₃ without the use of any stabilizers, Kang et. al, Chem. Mater., 8, 2209-2211, 1996. This is an iron (II) / iron (III) solution with a molar Ratio of 0.5 precipitated at pH 11-12 with the exclusion of oxygen, the <10 nm large Leads magnetite particles.

In addition to the areas of application mentioned above, magnetic colloids are also used in the area of medical diagnostics, analytics, molecular biological analytics and in Therapy used. To do this, the colloids with different functional Polymer substrates or proteins (matrix) coated, which have two basic functions have to fulfill. On the one hand, the coating should be as biocompatible as possible largely bypassing the reticulo-endothelial system (RES) the blood half-lives can be extended, on the other hand to the matrix bioligands z. B. in the form of Antibodies, antigens, receptors, lectins, oligosaccharides, amino acids, proteins or DNA / RNA fragments are chemically bound. This ligand coupling enables according the principle of affinity binding to the bioligands complementary analytes, such as. B. Antigens, antibodies, proteins, DNA fragments, cells, viruses or bacteria, their Determination in biochemical or molecular biological analysis, diagnosis and Therapy plays an important role. Matrices that have been described for such purposes are e.g. B. dextran, dextrin, albumin, silica gel, polystyrene, gelatin, polyglutaraldehyde, liposomes, Polyethyleneimine, polyvinyl alcohol, polyacrolein; they are from the PCT WO patents 90/07380, PCT WO 89/11154 and US Pat. Nos. 4,628,037, 3,970,518, 4,230,685, 4,654,267, 4,452,773, 4,369,226, 4,267,234, 3,652,761, 4,675,173 generally known. An overview of the various magnetic particles and their application are in "Scientific and Clinical Applications of Magnetic Carriers ", Haefeli et al. ed., Plenum Press, New York, 1997. Allen The aforementioned products have in common that they function from the complementary Interaction of a bioligand bound on the matrix with the one to be analyzed  Derive analytes. Your areas of application are thus limited to the known fields of Separation and analysis of biomolecules or the labeling of certain cells under Exploitation of the classic principle of affinity. All of the aforementioned magnetic particles common that they consistently have a low magnetic colloid content in the form of magnetite or iron oxides and thus have a low saturation magnetization, the one efficient heating of the particles as far as possible in an alternating magnetic field exclude and can therefore only be used in the usual analysis sectors.

In US Pat. Nos. 4,735,796 and 4,662,359, magnetic particles are described that are specific to the Tumor therapy can be used in such a way that those absorbed by the tumor tissue Magnetic particles inductively by means of an external high-frequency alternating magnetic field Temperatures above 40 ° C are heated and so to selectively die Tumor cells. The disadvantage of the above means and procedures for Tumor therapy, however, is that they have neither viable methods of coating with such Contain polymer substrates that have a clear coating with bioligands or Antibodies, as they are a prerequisite for use in diagnostic analysis, still demonstrate basic techniques for coupling the required biological ligands.

In contrast to the aforementioned means, the magnetic particles according to the invention can due to the combinatorial application of the separation principle and the inductive Heating for the first time for diagnostic, analytical and therapeutic purposes as well as for Decontamination can be used. This opens up the possibility of being biochemical and pre molecular biological analysis and medical diagnostics more efficiently than before shape.

The principle of action of the agents according to the invention is to apply a functional polymer matrix into which the inductively heatable magnetic colloids or fine disperse magnetic particles are encapsulated to bind biomolecules adsorptively or covalently are able to analyze such as. B. DNA / RNA sequences, antibodies, antigens, proteins, Cells, bacteria, viruses or fungal spores according to the complementary principle of affinity tie. After the analytes have been bound to the matrix, the magnetic particles can be combined in one high-frequency magnetic alternating field to those for analysis, diganostics and therapy relevant temperatures of preferably 40-120 ° C are heated.  

In order to make the aforementioned compounds and alloys relevant for bioanalytics To be able to heat temperatures requires a special design of the magnetic field in in relation to the magnetic field strength and the frequencies.

As a rule, current-carrying coils are used by one High frequency generator can be fed. Such coil systems and high frequency generators are general state of the art and are offered commercially. The dimension of the Coils depend on the respective sample sizes; they generally have a diameter of 1-10 cm and a length of 2-30 cm. The required output power of the HF Generators are usually between 0.5 and 1.5 kW. For heating up the magnetic samples basically two generator settings can be selected: a) high frequency in the range of 5-80 MHz and low magnetic field strength of 100-500 A / m or b) low frequency of 0.2-5 MHz in connection with a high magnetic field strength of 500-15000 A / m. Both Field parameter combinations ensure sufficient energy absorption by the Magnetic particles within a short application period (<5 minutes).

Because the energy absorption of the magnetic colloids and consequently the heating of the magnetic polymer particles of various substance parameters such as colloidal dispersity, Colloid composition, colloid concentration and the type of heat capacity of the Polymer matrix depends on the setting of a specific temperature Induction conditions can be adapted to the respective samples. So absorb bigger ones Magnetic particles consistently have less energy per mass than finer particles with the same Composition. Accordingly, for the inductive heating of coarsely dispersed colloids (<1 µm) high magnetic field amplitudes of <8 kA / m are necessary to achieve a significant To ensure energy consumption at a frequency of <1000 kHz. In contrast to For finely dispersed colloids with a particle size <200 nm, field strengths of 2-5 kA / m at a frequency of <1000 kHz for efficient heating. In addition to those on the Induction parameters to be adapted to the respective samples play the duration during the heating the induction for a given magnetic field setting and the coil design (coil radius, Length of the coil, coil turns) in relation to the volume of the sample to be treated an essential role.

In general, all compounds come as magnetic particles for the agents according to the invention in question, which can be inductively heated in an alternating magnetic field, d. H. the one have spontaneous magnetization. Compounds that have such properties in particular own, fall under the category of ferro, ferri and superparamagnets. Regarding the Classifications are based on the statements by Bean and Livington, J. Appl. Phys. 30, 1205,  1959 and Chagnon et al., U.S. Patent 4,628,037. This class of substances include e.g. B. all iron (II) and iron (III) oxides of the formula FeO, Fe₂O₃ and mixed iron oxides of the formula Fe₃O₄. Other connections, which are also ferromagnetic in nature and basically the The conditions of inductive heatability are nickel, cobalt, gadolinium and Rare earth metals dysprosium, holmium and terbium as well as ferromagnetic alloys non-ferromagnetic elements. Connections of this type, which are also called "Heusler alloys" are generally known, for example Composition Cu₂AlMn or Cu₂SnMn. Alloys made of iron, nickel, cobalt, Aluminum, copper, neodymium and boron, which because of their sometimes very high coercive force Hard magnetic materials are used, basically meet the above requirements Requirements. To the above-mentioned materials in the agents and To be able to use methods, it should preferably have a particle size of 0.0052-2 μm have one of 0.01-1 µm. This ensures that the magnetic colloids at the subsequent encapsulation by a functional polymer in finely dispersed form in the Polymer matrix are present.

The production of correspondingly finely dispersed magnetic colloids or ferrofluids is more general State of the art and generally takes place either by wet chemical precipitation processes or through a powder metallurgical process in which the magnetic particles are in a ball mill are ground over a longer period of time (see US Pat. Nos. 4,329,241, 4,827,945, 4,628,037, 3,917,538).

It has now been shown that inductive heating is made much easier as a result can choose ferromagnetic compounds that have a Curie temperature (hereinafter referred to as "Tc") of <250 ° C, preferably 40-120 ° C.

Ferromagnetic substances are particularly characterized in that their ferromagnetism passes directly into paramagnetism at the Curie point. This special property can now surprisingly to a quick and precise temperature setting of the Magnetic particles can be used by changing paramagnetic substances due to the drastic Decrease in magnetic susceptibility in the high frequency used here Do not let the alternating field area heat up inductively. Specifically, that means the temperature of the magnetic colloid oscillates exactly after a short heating-up period when the magnetic field is switched on around the Curie point, which is determined solely by the composition of the colloid. Under Applying this Curie point principle therefore plays the exact setting of the outside  Magnetic field as well as the duration of the induction within the stated principle required basic power and frequency largely a subordinate role.

The determination of the respective temperatures is primarily through the application in Determined within the framework of bioanalytics or medical application.

A number of compounds can be used as magnetic substances for the Tc range <120 ° C. These include mixed ferrites of the general formula Me _1-x Zn _x Fe₂P₄, where Me can be cobalt, nickel or manganese. By successively substituting the zinc with the aforementioned elements, all Tc in the range of 40-250 ° C can be covered.

Specifically for the temperatures of <90 ° C, 70-75 ° C and 40-60 ° C relevant in the field of DNA analysis and sequencing, corresponding to the denaturation of the DNA, the primer elongation reaction and the hybridization of the sequencing primer ( "annealing") the following mixed ferrites are suitable: for the range of <90 ° C Ni _0.73 Zn _0.27 Fe₂O₄ or Co _0.45 Zn _0.55 Fe₂O ₄ , for the temperature range 70-75 ° C Co _0.62 Zn _0.38 Fe₂O₄ and for the range 40-60 ° C z. B. Ni _0.75 Zn _0.25 Fe₂O₄, Co _0.39 Zn _0.61 Fe₂O₄ or Mn _0.4 Zn _0.38 Fe ₂ O ₄ . Other compounds that meet the conditions required above are: Li-Fe-Cr oxides of the general composition Fe _x Li _y Cr₂O₄ (x = 0.1-0.9, y = 0.5), chromium spinels such as MnCr₂O₄ (Tc : 77 ° C) and MnFe _2-x Cr _x O and MgCrFeO₄ (Tc: 50 ° C). In principle, there are also rare earth / transition metal alloys from the 2:17 series, especially Pr₂Fe₁₇ and Er₂Fe₁₇. A fine adjustment in the direction of higher Tc can be carried out in such a way that part of the iron can be replaced by cobalt. Also known as the Heusler alloys from z. B. manganese, tin, arsenic, antimony, boron, bismuth with additions of copper meet the required conditions.

Such compounds or alloys with low Tc are produced by wet chemistry with the help of the co-precipitation of an appropriate saline solution, spray drying, the Freeze drying, the sol-gel process or the precurser method. Alternatively, you can these connections can also be produced by a grinding process in a ball mill lasting several days. Both methods of production as well as the adjustment of the particle sizes of the aforementioned Connections are general state of the art (cf. R. Valenzuela, "Magnetic Ceramics", Cambridge University Press, 1994, J. Smit and H.P. J. Wijn, "Ferrits", Wiley, New York, 1959).

In addition to the magnetic properties of the colloids and the resulting Induction behavior, the type of polymer matrix plays a role in the application of the magnetic particles which are encapsulated play an essential role. The polymer matrix must do the following Fulfill conditions: a) chemical functionality and b) biocompatibility. The chemical Functionality enables the chemical coupling of bioligands e.g. B. in the form of  Cell receptors, antibodies or nucleic acid fragments according to the known methods for Binding of ligands to carrier media. Biocompatibility meets the requirements for the Use of the magnetic particles primarily in the diagnostic and therapeutic area.

Polymers that meet these requirements usually belong to the class of hydrogels whose biocompatibility is primarily derived from the high water content (<80%). Basis for determining biocompatibility, which is not accurate in the biochemical field is defined, the cytotoxic properties of the encapsulated magnetic colloids. To the magnetic particles are exposed to HeLa and endothelial cell suspensions over a period of 24 hours incubated at 37 ° C. After incubation, the cell integrity and Cell morphology by staining with ethidium bromide / acridine orange and with the help microscopic methods determined (see Warring, Antibiotics III 141, Springer Verlag, 1975, Darzynkiewicz and Kapuscinski, Flow Cytometry and Sorting, Melamed et al. Ed., Wiley, P. 291, 1990). As a requirement for the selection of the relevant polymer matrices, a <95% maintenance of cell integrity and cell morphology used as a basis. Polymers that fulfill the above criteria, are preferably poly- and oligosaccharides, polysaccharide Derivatives, such as hyaluronates, alginates, dextran, agarose, dextrin, starch, heparin, Hydroxyethyl cellulose, further synthetic polymers such as polyvinyl alcohol, polyacrylic acid, Polyurethanes, polyoxyethylene, polylactide, polyglycolide, polycyanoacrylates, Polyhydroxyethyl methacrylate and copolymers of these substances. Proteins also come such as B. gelatin, casein, collagen, albumin, fibrinogen or polyamic acids in question.

To encapsulate the magnetic colloids with the polymers, there are basically those Methods applicable which are able to completely encapsulate the magnetic colloids and result in appropriately isolated nano- or microparticulate magnetic particles. Advantageously come the generally known methods such as suspension crosslinking ("suspension crosslinking "), emulsion polymerization, suspension polymerization or precipitation process ("Solvent Evaporation Process") from the organic phase. The Differentiation between emulsion and suspension in the following refers to the Different particle sizes: emulsion for particle sizes <1 µm and suspension for those <1 µm.

In the suspension crosslinking process, an aqueous polymer solution in which the magnetic colloid is dispersed in a water-immiscible phase (organic Phase) suspended and then with a suitable bifunctional or trifunctional crosslinker networked (see US Pat. No. 4,345,588 and PCT application EP 96/0239). Water-soluble polymers which are preferably suitable for this process are e.g. B. serum albumin, proteoglycans, Glycoproteins, polyvinyl alcohol, cellulose, agarose, dextran, alginates, hyaluronates, starch, Polyhydroxyethyl methacrylate. Suitable organic phases are oils such as paraffin oils, Vegetable oils, silicone oils, cottonseed oil or organic solvents such as chloroform, Butanol, heptanol, toluene, benzene, ethyl acetate, hexane, heptane, octane and mixtures the same. To determine the droplet size in the suspension process in the direction of that for the preferred small particle sizes of 0.5-5 µm for analytical purposes the dispersion phase usually 0.3-15 wt .-% stabilizers, which preferably belongs to the group of Polyoxyethylene adducts, polyoxyethylene sorbitol esters, polyethylene propylene oxide Block copolymers, alkylphenoxypolyethoxyethanols, fatty alcohol glycol ether Phosphoric acid esters, sorbitan fatty acid esters, polyoxyethylene alcohols, polyoxyethylene sorbitan fatty acid esters, or belong to polyoxyethylene acids added. The volume ratios of organic phase to polymer / magnetic colloid phase vary between 10: 1 and 60: 1, that of the polymer phase to the colloid phase generally between 1: 0.5 and 1: 4, preferably between 1: 1 and 1: 3, whereby the polymer / magnetic colloid ratio by the respective Amount of solid content in the colloid is determined. This is usually between 1 and 10% by weight of the solids content in the polymer is advantageously 20 to 60 % By weight. This high magnetic colloid content, through which the agents according to the invention fundamentally different from the conventional magnetic polymer carriers chosen because, in addition to the magnetic susceptibility, the efficiency of the inductive Heating, d. H. the heating or heating achieved under given induction conditions Heating rate is determined directly by the amount of magnetic content in the particles. Suitable crosslinkers for the polymer matrix are difunctional and trifunctional substances which are compatible with the functional groups of the matrix (z. B. hydroxyl, carboxyl, amino groups) can react. Suitable examples for this are: alkyl dihalides, glutaraldehyde, di-  and triisocyanates, divinyl sulfone, cyanogen bromide, triallyl isocyanurate, divinyl ethylene urea, Epichlohydrin, 2-chloro-1-methylpyridinium iodide, 1,4-butanediol divinyl ether, divinyl succinate, Divinyl glutarate, divinyl adipate. The amount of crosslinker, primarily due to the fineness of the magnetic colloid is usually 0.5-10 mol%, based on the Polymer content. Consistently higher levels of crosslinking are required in the case of very fine Colloids to prevent diffusion out of the matrix.

Analogous to the crosslinking process in suspension, suspension polymerization also offers the Possibility to encapsulate the magnetic colloids in a defined way. To do this, a  water-soluble vinyl monomer in an organic immiscible with the monomer Phase suspended and then radical with the addition of suitable stabilizers polymerized. Methods of this type are known from the literature (cf. Paine et al., Macromolecules, 23, 3104, 1990, Tuncel et al., J. Appl. Polym. Sci., 50, 303, 1993, Takahashi et al., J Polymer Sci., Part A: Polymer Chem., 34, 175-182, 1996). Coming as monomers water-soluble monomers such. B. vinyl pyrrolidone, acrylamide, 2-hydroxyethyl methacrylate, Hydroxyethyl acrylate, acrylic acid, methacrylic acid and mixtures thereof in question. The Dispersion phases consistently correspond to those given for suspension crosslinking organic phases that are not miscible with water. As suspension stabilizers come in the Usually the same stabilizers for use as in the aforementioned crosslinking process. Within this process technology, 2-hydroxyethyl methacrylate has become the main encapsulation proven to be advantageous since the polymer on the one hand meets the requirements of a Hydrogels fulfilled, on the other hand is highly functional due to its chemical structure. For the Encapsulation with this polymer has surprisingly been found in the form of stabilizers synthetic polymer systems such. B. polymethyl methacrylate, methacryloyl end groups containing polymethyl methacrylate, polystyrene, polystyrene derivatives or polybutadiene derivatives proven to be particularly advantageous. The proportions of polymer to magnetic colloid are analogous to those in the aforementioned networking process.

Another method for encapsulating the magnetic colloids is derived from the known Process for the preparation of polymer particles by means of emulsion polymerization as they for styrene, methyl methacrylate and glycidyl methacrylate (see Woods et al., J. Paint Techn., 40, 541-548, 1968, Smigol et al, Angew. Macromol. Chem., 195, 151-164, 1992, Okubo and Shiozaki, Polymer Int., 30, 469-474), 1993). This will advantageously either by adding anionic surfactants such as polyacrylic acid or dodecylbenzyl sulfonate, nonionic Surfactants such as alkylphenyl polyethylene glycols or polyethylene glycol trimethylnonyl ether, combined addition of anionic and non-ionic surfactants or under emulsifier-free conditions carried out, the latter procedure optionally in Presence of sodium chloride can take place.

Using this process technology as a basis, it has now surprisingly been possible to show that magnetic colloids stabilized by the addition of lipophilic or semi-lipophilic surfactants encapsulated in the polymer particles which form for the emulsion polymerization batch become. Suitable surfactants are those that have an HLB value (Hydrophilic-lipophilic balance, see Griffin, J. Soc. Cosmetic Chemists, 5, 249-255, 1954) have from 1-12. Examples include: polyethylene propylene oxide block copolymers,  Alkylphenoxypolyethoxyethanols, polyoxyethylene alcohols, polyoxyethylene sorbitan fatty acid esters, Sorbitan fatty acid esters.

The following reaction batches have proven to be suitable: monomer concentration 0.7-5.2 mol / liter, Initiator concentration 1-10 mmol / liter, stabilizer concentration 0.1-80 mmol / liter; the sodium chloride concentration can optionally be up to 20 mmol / liter. This Approaches provide magnetic particles with particle sizes between 0.4 and 8 µm.

To produce the magnetic particles using the solvent evaporation process, which is a special As a method within precipitation technology, one generally starts from one Two-phase system consisting of a water-immiscible organic polymer phase and an aqueous dispersant. The lipophilic polymer phase in which the magnetic colloid is first dispersed in the aqueous under defined stirring conditions Phase suspended. The polymer falls through contact with the aqueous phase Evaporation of the solvent in the form of isolated particles. Experience has shown that With the help of the stirring speed as well as by adding surface-active substances both for the organic as well as the dispersion phase the particle sizes in the range of 1-250 µm can be varied. Alternatively, the organic polymer phase can also be carried out using a Enter the spray device in the hydrophilic phase. Spray systems and processes of this type are general state of the art (cf. Iwata and McGinity, J. Microencapsulation, 9, 201-214, 1992; Thoma and Schluetermann, Pharmazie, 47, 115-119, 1991). Polymers that are suitable for a Such manufacturing processes are basically those polymers that are used in organic Solvents, but not soluble in an aqueous medium. Examples include: polystyrene, Poly (lactide-co-glycolide), polymethacrylate, polyamides, polyester, polyvinyl ether, Polyvinyl acetals, polyvinyl ketones, polyvinyl halides, polyvinyl esters or polycarbonates. Experience has shown that suitable solvents for these polymers are, for. E.g. dimethylformamide, Dimethyl sulfoxide, trifluoroacetic acid, trifluoroethanol, phosphoric acid tris dimethyl amide, dioxane, Tetrahydrofuran, esters, pyridine, halogenated hydrocarbons and aromatic, halogenated and non-halogenated compounds. The volume ratios of the aqueous phase to the organic polymer phase, which usually contains 1-10% by weight of polymer consistently 10-30: 1.

The reverse procedure, a polymer soluble in an aqueous medium Dispersion in a lipophilic phase (preferably oils or chlorinated hydrocarbons) and then adding an organic, water-miscible non-solvent (e.g. Precipitation of acetone, methanol, ethanol) leads in analogy to the aforementioned procedure also for defined colloid encapsulations. Suitable polymer systems in question come, are 1-10% aqueous solutions of, for example, polyvinyl alcohol,  Polyacrylic acid, polysaccharides, polysaccharide derivatives, polyhydroxyethyl methacrylate or Proteins.

The coupling of the ligands relevant for bioanalytics such as oligosaccharides, glycoproteins, Avidin, biotin, lectins, antibodies, antigens, DNA or RNA fragments to the polymer-coated magnetic colloids are carried out according to the known methods of immobilization of biological ligands to polymeric supports (cf. "Methods in Enzymology", Mosbach ed., Vol 135 Part B, Academic Press, 1987, pp. 3-169). Such agents come in as coupling media Question that has proven itself in the preparation of affinity resins such as B. cyanogen bromide, Carbodiimide, hexamethylene diisocyanate, tosyl chloride, tresyl chloride, 2-fluoro-1-methyl pyridinium toluene-4-sulfonate, epichlohydrin, N-hydroxysuccinimide, chlorocarbonate, isonitrile, Hydrazide, glutaraldehyde, 1,1'-carbonyl-diimidazole, 1,4-butanediol diglycidyl ether. Especially In addition, the coupling of the bioligands via so-called spacer molecules is favorable, hetero-bifunctional, reactive compounds that interact with both the functional groups of the Matrix (carboxyl, hydroxyl, sulfhydryl, amino groups) as well as with the bioligand can form a chemical bond. Examples include: succinimidyl-4- (N-maleiimidomethyl) - cyclohexane-1-carboxylate, 4-succinimidyloxycarbonyl-α- (2-pyridyldithio) toluene, Succinimidyl 4- (p-maleimidophenyl) butyrate, N-γ-maleimidobutyryloxysuccinimide ester, 3- (2- Pyridyldithio) propionyl hydrazide, sulfosuccinimidyl-2- (p-azidosalicylamido) ethyl-1,3'- dithiopropionate (cf. Ullmann's Encyclopedia of Technical Chemistry, 4th ed., vol. 10, and G.T. Hermanson, "Bioconjugate Techniques," Academic Press, San Diego, 1996).

From the combinatorial principle on which the agents according to the invention are based Affinity binding and inductive heating open up applications whose breadth the conventional means remains closed. This mainly affects the Decontamination of pathogens or infectious germs in biological fluids (e.g. Blood, plasma, serum, stored blood). Viruses, e.g. B. hepatitis viruses or that "Human Immunodeficiency Virus" (HIV), in addition bacteria, fungal spores or animal proteins, which in the course of a vaccine serum development also coincide and increase Allergic reactions can result in selective removal by following the magnetic particles Binding of the pathogens are inductively heated to preferably 70-100 ° C, which the kill the affected germs or inactivate them as far as possible. In the field of medical Therapy also gives the opportunity to add medication to the polymeric magnetic particles miteinkapseln ("drug depot"), which, administered in the body, by inductive heating  can be released into the body in a controlled manner by applying the polymer matrix Temperatures above the glass temperature are heated, from which a reinforced Drug release results.

The essential feature of the agent according to the invention is the encapsulated Magnetic colloids with the help of an external magnetic field to defined temperatures in the area heating preferably from 40-120 ° C. The goal here is to determine certain on the polymer matrix immobilized biomolecules or bioligands in the context of special implementations or to convert bioanalytical processes into an active form that is not at normal temperature or can only be realized to a limited extent. Examples of such implementations or applications, which take place or are possible at higher temperatures are the isomerization of glucose Syrup to glucose and fructose using glucose isomerase, a reaction that is noticeable only at 60 ° C and DNA sequencing using Taq DNA polymerase, a highly processing enzyme of the bacterium thermus aquaticus, which only becomes active above 70 ° C. Another example is the separation of DNA double strands at <90 ° C (denaturation), a method that is also used for DNA sequencing e.g. B. in the context of medical Diagnostics for the determination of pathogens (viruses, bacteria, fungal spores) plays an increasingly important role.

Due to the combination of an affine bond and inductive heating, the surprisingly use the agents according to the invention in the clinical-medical field, at z. B. eliminate pathogenic germs in potentially infectious substances. To this end receptor molecules or bioligands are coupled to the magnetic particles Pathogen can enter into a complementary bond. After binding the pathogen to the Magnetic particles become inductive to them in an external, high-frequency magnetic field Temperatures heated up that can kill the respective infection germ. Through the Specific binding magnetic particle pathogen ensures that the heating on the Magnetic particles and consequently on the pathogens bound by the magnetic particles Substances remains limited. The remaining components of the liquid become practical not heated up. The aforementioned procedure can be used, for example, for elimination certain viruses in the blood or stored blood, e.g. B. hepatitis viruses, HI viruses (Human Immunodeficiency Virus) can be used. For this purpose, virus-specific antibodies are sent to the Magnetic particles chemically coupled. The magnetic particle-pathogen complex then becomes inductively heated to 80-120 ° C for short time intervals (<one minute), a treatment which is used for extensive inactivation of the corresponding viruses is suitable.  

Another area of application for the inductively heatable magnetic particles is the field of separation technology, in particular affinity separation technology. In this type of separation, in addition to the selection of suitable affinity ligands, which determines the selectivity of the process, the elution behavior of the affinity medium also plays a decisive role in the efficiency of the separation. As a rule, certain buffer solutions are often added with the addition of a chaotropic salt, e.g. As sodium rhodanide, the molarity and pH values of which must be carefully selected in order to enable a largely complete elution of the analyte while maintaining the biological activity. The use of the magnetic particles according to the invention surprisingly opens up a new perspective here of making the elution of the analytes much more efficient. As a result of the inductive heating of the magnetic particles, there is a weakening of the bioligand analyte bond, which requires the analyte to be easily detached from the stationary phase while avoiding the otherwise rigid elution conditions. This novel elution principle can be used particularly advantageously in the field of avidin or streptavidin-biotin technology. The high affinity between avidin and biotin is increasingly used in biochemical and molecular biological analysis, for example in the field of DNA sequencing, DNA detection, hybridization assay, cell separation or enzyme assay. Here, the extremely high affinity bond between avidin and biotin (K _d <10 ^-15 ) is used in such a way that biotinylated analytes z. B. in the form of DNA probes or oligonucleotides that can be easily isolated from a mixture of substances. The fundamental problem with all avidin-biotin separations is that, owing to the extremely high binding constants, elution of the biotin analyte is practically impossible without deactivating the avidin and the analyte. It has now surprisingly been found that the induction heating of the avidin-coupled magnetic particles makes it possible to elute the biotinylated analytes. For this purpose, the magnetic particles are generally inductively heated to 50-70 ° C. and simultaneously with a 0.1 M glycine-NaOH buffer, pH 8.5-10, containing 0.5-1 M NaCl and / or 40-60% ethylene glycol. B. eluted.

In a direction analogous to the elimination of pathogens, another application is aimed at: is about obtaining vaccine sera, which are obtained from animals, in a higher purity. The The general aim is to use the antisera obtained after the animals have been immunized largely free of animal accompanying substances such as antibodies, which are potentially allergenic can work. To remove specifically animal antibodies, the Fc fragment of the  Antibody-directed secondary antibodies coupled to the magnetic particles. After appropriate incubation of the anti-Fc-IgG coated magnetic particles over a period of time of, depending on the serum volume, 15-30 minutes the magnetic particles are in intervals (<one Minute) inductively according to the inventive method over a total period of 10 Minutes heated to temperatures of 60-80%. This treatment is usually sufficient to inactivate <90% of minor components of animal origin.

The agents and methods according to the invention are described below with the aid of a few examples explained in more detail.  

example 1

With 4.5 g vinyl acetate, the 0.01 g polyethylene oxide (molecular weight 3000), 0.15 g triallyl isocyanurate, 0.05 g of benzoyl peroxide and 3 ml of magnetite colloid DHYS1 (Liquids Research, Wales) were added are in 50 ml of water containing 25 wt .-% NaCl, under nitrogen atmosphere for 12 h 60 ° C carried out a suspension polymerization. Magnetic, pearl-shaped polymers fall with a particle diameter of 4-8 µm and a magnetite content of 32% by weight. The After a corresponding centrifugation step, the product is washed several times with water. The Subsequent hydrolysis to polyvinyl alcohol for 20 hours is carried out using 20% NaOH Room temperature, which is followed by several washing steps with water. 0.1 g of the resulting dried product are mixed with 2 ml of 3 M NaOH and 1 ml of epichlorohydrin and at 2 h 60 ° C activated. Then the product is alternated with water and acetone several times washed; this is followed by drying in a vacuum. The epoxy equivalent is 183 µmol / g Carrier (determined according to Sundberg and Porath, J. Chromatogr., 90, 89, 1974). 0.05 g of Epoxy carriers are mixed with 0.5 ml 0.1 M bicarbonate buffer, pH 9.8, in the 10 µg DNA (850 bp), the methods previously described according to a method described by Chu and Organ, DNA, 4, 327, 1985, on the 5'- End amino-modified, incubated at 35 ° C for 24 h. The sample is then washed several times with water. The coupling yield is 43% (photometric in Supernatant measured). To the coupled DNA according to the known methods (see "Advances in Biomagnetic Separation ", Uhlen et al. Ed., Eaton Publishing Co., 1994) for genetic analysis to be able to use the magnetic particles in a magnetic field (5 coil turns, Coil diameter 5 cm, coil carrier 12 cm) at a frequency of 500 kHz and a magnetic field strength of 11 kA / m inductively heated for 2 minutes. The hybridized immobilized DNA is thereby separated into the two double strands, which are created of a neodymium-iron-boron hand magnet can be separated. The further analysis steps are carried out in accordance with the known methods for DNA or gene analysis.

Example 2

A mixture consisting of 45 ml of 2-butanol and toluene (55: 45 wt .-%), 9.6 ml under Nitrogen-distilled 2-hydroxyethyl methacrylate, 0.95 g polymethyl methacrylate (MW 2.2 × 10⁵), 4.2 ml magnetite colloid stabilized with polyvinylpyrrolidone (MW 20,000) (prepared according to Tiefenauer et al., Bioconjugate Chem., 4, 347-352, 1993) and 0.18 g of 2,2'-azobis (isobutyronitrile) is vented through a stream of nitrogen over a period of 30 minutes.  The mixture is then mixed with ice-cooling and under a nitrogen atmosphere for 4 minutes Ultrasonic treated (Bandelin Sonoplus homogenizer GM 200, 200 W, 20 kHz). After that the polymerization is carried out with stirring (250 rpm) at 70 ° C. over a period of 7 Hours. The polymer is then magnetically separated from the supernatant and repeated several times Toluene, acetone and finally washed with water. Polymer beads with a drop Grain size of 3-5 µm; the magnetite content is 36% by weight. A 40 wt .-% aqueous Suspension of these particles is in a magnetic field according to Example 1 in four minutes at 65 ° C. heated up.

0.2 g of the product dried over P₂O₅ in a vacuum with one ml of absolute Dimethylformamide, in which 0.6 mmol of 4-dimethylaminopyridine and 0.5 mmol of 2-fluoro-1-methyl pyridinium toluene-4-sulfonate, are dissolved, added and activated for 45 min at room temperature. The Product is washed several times alternately with dimethylformamide and acetone as well twice with 0.05 M bicarbonate buffer, pH 9.2. 100 µg anti-tumor necrosis factor α (TNFα) Antibodies, dissolved in one ml of the wash buffer, are then incubated over a Bound to the support for a period of 24 h at room temperature. Coupling yield 64%. For Deactivation, the carrier is washed several times with 0.1 M Tris-HCl buffer, pH 8.4 and then incubated for 12 h in 0.1 M Tris-HCl buffer / 5% glycerol, pH 7.5.

The carrier thus obtained is used to inactivate TNFα, a pathogen that is a strong one Mediator for septic shock is used by the carrier with blood or serum in it Is brought in contact and after formation of the TNFα through one-minute induction intervals a period of 10 min. is heated inductively.

Example 3

0.1 g of inductively heatable magnetic particles, which were produced according to Example 2, with 0.1 g of bromcaproic acid, which is dissolved in 2 ml of 3 N NaOH, reacted at 70% for 2 h. It follows repeated washing with water with the help of a magnetic one Separation step. The support is then washed twice with 0.005 M HCl and then with 1 ml of 0.1 M morpholino-ethanesulfonic acid buffer (MES), pH 4.2, in which 20 mg N-Cyclohexyl-N '- (2-morpholinoethyl) carbodiimide-methyl-p-toluenesulfonate are dissolved for 30 min. incubated. After activation, the carrier is washed five times with ice water and then with a ml of 0.05 M bicarbonate buffer, pH 8.6, the 0.03 mg of dissolved streptavidin  contains, incubated for 24 h at room temperature. The coupling yield 31% (determined) according to Lowry et al, Biol. Chem., 193, 2654, 1951).

The carrier obtained in this way is capable of producing biotinylated DNA single or double strands bind, which are used for the sequencing according to the known methods in gene analysis can.

Example 4

2 ml of a 20% by weight aqueous gelatin solution are mixed with 150 mg of Ni _0.24 Zn _0.76 Fe₂O₄ (particle size 30-80 nm, Tc = 75 ° C) and the dispersion in 60 ml of 5% by weight containing sorbitan monooleate containing toluene / chloroform mixture (40: 60 wt .-%). After by introducing a stream of argon for 10 min. the atmospheric oxygen was removed, the mixture is cooled with ice for 2 min. treated with ultrasound according to Example 2 and then introduced into a 350 ml pre-cooled toluene / chloroform / sorbitan monooelate mixture corresponding to the above composition with stirring (1000 rpm). The gelatin-ferrite droplets are crosslinked by subsequently adding 50 ml of toluene saturated with glutaraldehyde. The crosslinking is carried out with stirring at 0 ° C. over a period of 4 h. Then it is washed several times with isopropanol, 50% aqueous acetone solution and finally three times with 0.1 M potassium phosphate buffer pH 7.5. Magnetic particles with a particle size of 2-5 μm and a magnetic colloid content of 33% by weight are obtained. The particles obtained can be heated to a constant 75 ° C. in the magnetic field described in Example 1 within one minute.

Activation of the magnetic particles with 2 ml 0.1 M potassium phosphate buffer, pH 8, the 12% Contains glutaraldehyde, takes place for 4 h at room temperature, which involves intensive washing processes Connect water for about an hour.

Carriers activated to the glutaraldehyde can be counteracted by the known methods Virus-directed antibodies by incubation for several hours in a 0.05 M potassium phosphate Buffer, pH 7.5-8.5, are covalently bound. Inductive heating of the magnetic particles over a period of 10 min. at 75 ° C leads to inactivation of the viruses.

Example 5

140 mg of a nickel-zinc ferrite with the composition Ni _0.73 Zn _0.27 Fe 2 O 4 (particle size 40-70 nm, Tc = 95 ° C.) are introduced into a gelatin suspension prepared according to Example 4 and then crosslinked with glutaraldehyde. The magnetic particles thus obtained can be heated to a constant 95 ° C. within one minute under the magnetic alternating field conditions set out in Example 1. 50 mg of the carrier thus obtained are reacted with 12% glutaraldehyde solution according to the above example and then washed with water. 5 ug at the 5 'end amino-modified DNA, which was prepared according to Example 1, are coupled to the activated carrier by incubation for ten hours at 30 ° C. The yield is 46%.

The immobilized DNA double strands can be induction at 95 ° C for three minutes separate according to Example 1 into the single strands, which then the conventional, further Genetic analysis are accessible.

Example 6

One by Pulfer and Gallo (In: "Scientific and Clinical Application of Magnetic Carriers", Häfeli  et al. Ed., Plenum Press, New York, p. 445, 1997) of magnetic carriers is used in a modified manner as follows: 4 ml of a 25 wt .-% aqueous dextran solution (average molecular weight 40,000) with 1.2 ml of 10 M NaOH and 3 ml of an aqueous MnZnFerrit emulsion (BASF, particle size approx. 12 nm) added. The mixture is for 2 min. according to Example 2 under ice cooling with ultrasound treated. The mixture is then made into a conventional 80 ml syringe Cottonseed oil containing 0.3 g polyoxyethylene sorbitan monooleate is injected and again for 3 min. sonicated at 0 ° C. With stirring (2000 rpm), 200 ml of ether, in the 4 g of cyanogen bromide are added. After 15 min. the magnetic particles become short centrifuged and washed several times alternately with ether and ethanol. The won Particles have a size of 2-5 µm and a magnetic component of 26% by weight. By Applying the alternating magnetic field according to Example 1, the magnetic particles can within from 2 min. be heated to temperatures <100 ° C.

0.1 g of the dextran particles obtained are then mixed with 2 ml of 0.2 M bicarbonate buffer, pH 11.3, in which 10% cyanogen bromide are dissolved, for 15 min. activated. After washing several times Water and the activation buffer, the carrier for 10 h in 2 ml of 0.2 M bicarbonate buffer, pH 9.0, which contains 20 wt .-% aminocaproic acid, incubated at 4 ° C. The multiple times with water washed, carboxylated carrier is incubated twice in 0.005 M HCl, once with water washed and then with 2 ml of 20 mg of N-cyclohexyl-N '- (2-morpholinoethyl) carbodiimide methyl  0.1 MES buffer pH 4.2 containing p-toluenesulfonate for 30 min. at room temperature activated. It is washed five times with ice water, followed by a 24 hour incubation a 0.05 M phosphate buffer, pH 8.5, containing 2 ml of 10 μg streptavidin. The coupling yield is 67%.

The carrier obtained in this way binds biotinylated DNA fragments, which are removed using a 1 M NaCl containing 0.1 M glycine-NaOH buffer, pH 9.0 in combination with repeated, short inductive heating (<1 min.) can be eluted largely quantitatively.

Example 7

0.05 g of dextran carrier prepared according to Example 6 are mixed with cyanogen bromide and N-cyclohexyl N '- (2-morpholinoethyl) carbodiimide-methyl-p-toluenesulfonate according to the above representation activated and with 1 ml 0.05 M phosphate buffer, pH 8.5, in which 10 µg one against the bacterium Mycobacterium tuberculosis-directed antibody are dissolved, incubated at 4 ° C. for 30 h. The Coated carriers are able to bind tuberculosis bacteria, which are subsequently caused by a three minute induction can be inactivated according to Example 1.

Example 8

20 ml of a 2% aqueous Na alginate solution (MW 70,000) containing 0.1% polyvinyl alcohol (MW 22,000) are mixed with 150 mg of magnetite (particle size 8-15 nm), which according to the instructions of Kang et al. (Chem. Mater., 8, 2209, 1996) were prepared, added and 3 min. treated with ultrasound under ice cooling. After a two-minute interval with constant cooling, 50 µg insulin (MW 5777, 25 U / mg) are added to the suspension, which is then again for two minutes. is sonicated with cooling. The mixture is then introduced into 220 ml of silicone oil (viscosity _{20 ° C} .: 100 mPa.s) in which 10% by volume of polyethylene-polypropylene block copolymer (Pluronic® 6200) are dissolved, with brief stirring, and then three times for one minute each sonicated at one minute intervals with constant cooling. The mixture is then stirred vigorously (3000 rpm). After 10 seconds, 40 ml of a 0.2 M CaCl₂ solution are added dropwise and the suspension is stirred for a minute after the addition of the CaCl₂ solution. The polymer carrier is then separated from the dispersing solution by centrifuging briefly. This is followed by repeated washing with a 10% NaCl solution and a phosphate-buffered saline solution, pH 7.2. Magnetic particles with a size of 4-12 μm are obtained, which have an insulin encapsulation yield of <60%. A 50 wt .-% aqueous suspension of the particles can be within 10 min. heat to temperatures of <50 ° C in the high-frequency field described in Example 1.

The magnetic particles can be used as a medication depot Inductive heating up the insulin release depending on the temperature and heating time accelerates the factor 1.5-2.

Example 9

10 ml of a 10% starch solution are mixed with 350 mg polyvinylpyrrolidone (MW 15,000) stabilized MnCr₂O₄ (particle size 30-85 nm, Tc = 77 ° C) and 3 min. With Ultrasound treated according to Example 2. The mixture is then in 100 ml at 80 ° C. preheated vegetable oil containing 4 ml of a polyethylene polypropylene block copolymer (Pluronic® 6100) contains, stirred in (2000 rpm). After 2 min. the mixture is made using an ice bath cooled with stirring. Solid 1-4 µm particles with a magnetic content of 31% fall by alternately suspending in ice water and 50% aqueous acetone solution be washed several times. The product dried in vacuo is then 3 ml Epichlorohydrin and 5 ml 4 M NaOH crosslinked at 55 ° C for 2 h. This includes intense alternate washing with water and aqueous acetone solution. For remaining oxirane groups to remove, the carrier is incubated for 24 hours in 0.1 M Tris-HCl buffer, pH 8.5. After washing several times with water and 0.1 M bicarbonate buffer, pH 11.3 100 mg of the magnetic particles previously dried in a vacuum by reaction for 15 minutes 30 mg cyanogen bromide in 2 ml 0.1 M bicarbonate buffer, pH 11.3, activated at room temperature. After Repeated washing with the activation buffer and water is done by twelve hours Incubate 1.5 ml containing 20 µg anti-CEA antibody (Carcinoembryonic Antigen) 0.1 M bicarbonate buffer, pH 8.5, obtained at 4 ° C a carrier that is able to CEA specifically bind supporting tumor cells. By induction heating for four minutes in accordance with Example 1, the tumor cells can be completely inactivated.

Claims (14)

1. Magnetic particles consisting of an inductively heatable magnetic core and one polymeric matrix in which the magnetic core is completely encapsulated and attached to the ligands are chemically coupled. 2. Magnetic particles according to claim 1, characterized in that the magnetic core there is inductively heatable nanoparticulate or microparticulate magnetic particles, that make up at least 20% by weight of the magnetic particle. 3. Magnetic particles according to claims 1 and 2, characterized in that the magnetic Core made of ferromagnetic, ferrimagnetic or superparamagnetic colloids or Suspensions exist. 4. Magnetic particles according to claims 1 to 3, characterized in that the inductive heated core consists of double metal oxide / hydroxides or magnetite. 5. Magnetic particles according to claims 1 to 3, characterized in that the inductively heatable core is a ferrite with a Curie temperature of 40-250 ° C, which has the general formula Me _1-x Zn _x Fe₂O₄, in which Me is iron, Can be cobalt or nickel. 6. Magnetic particles according to claims 1 to 5, characterized in that the polymeric matrix from polysaccharides or polysaccharide derivatives, alginic acids, alginic acid esters, There is hyaluronic acid, hyaluronic acid esters, cellulose, starch, agarose, dextran, dextrin. 7. Magnetic particles according to claims 1 to 5, characterized in that the polymeric matrix consists of natural proteins, modified proteins or polyamino acids. 8. Magnetic particles according to claims 1 to 5, characterized in that the polymeric matrix from a synthetic hydroxyl, aldehyde, amino or carboxyl groups Polymers or copolymers.   9. Magnetic particles according to claims 1 to 8, characterized in that the polymer Matrix antibodies, oligosaccharides, blood group antigens, oligonucleotides, polynucleotides, Enzymes, avidin, streptavidin, biotin, cell receptors, antibodies against cell receptors, Cell surface markers, lectins or glycoproteins are chemically bound. 10. Magnetic particles according to claims 1 to 8, characterized in that the polymer Matrix polymerases are bound. 11. A method for producing inductively heatable magnetic particles according to claims 1 to 9, characterized in that nanoparticulate or microparticulate magnetic particles into one polymer matrix by means of emulsion polymerization, suspension polymerization, Precipitation method ("solvent evaporation technique") or suspension crosslinking are encapsulated and reactive groups coupling to their surface with bioligands be generated. 12. Use of inductively heatable magnetic particles according to one or more claims 1 to 10 for the analysis of nucleotide sequences. 13. Use of inductively heatable magnetic particles according to one or more claims 1 up to 10 for the separation and inactivation of cells, viruses, bacteria or fungi from blood, Plasma, serum or blood supplies. 14. Use of inductively heatable magnetic particles according to one or more claims 1 to 10 as a drug depot in medical therapy.