POLYMER COATED METAL SURFACES

Abstract: A solid phase can be coated superficially with a polymeric coating by substituting immobilized metal ions on the surface of the solid phase with a hydrophilic polymer or a derivative or aggregate thereof, through a chemisorption process. The solid phase may comprise a magnetic material such as magnetite, and may have a particle form. The coating can be produced by bringing the solid phase into contact with a solution of a reagent which includes at least one metal chelating and one reactive group, and subsequently bringing the solid phase into contact with a solution of a compound which reacts with the chelater reagent absorbed on the solid phase. The polymer layer may constitute a polyhydric alcohol, a polyamine, or a polyamide, for exam­ ple a polysaccharide, such as agarose, a protein or a peptide, a polyacryl amide, etc. and the reactive group may be a vinysulphone or an epoxide, an aminothiol or a hydroxyl. The metal-chelate forming group is preferably a carboxy-­ methylated amine and the soluble compound is a water-­ soluble polymer, such as dextran, polyethylene glycol, dextran or some other polysaccharide.

Description: A novel method of coating the surface of a metal or insoluble metal compound with a monomolecular polymeric layer is IMA-technique ( I mmobilized M etal Ion A ffinity) and is based on the ability of metals and insoluble inorganic compounds that contain metal ions at the phase boundary to combine with a polymer that has been substituted with a metal chelate builder. The invention concerns a product for the separation of biological molecules and particles, consisting of a solid phase with an internal core such as tube, particle etc. with a surface containing immobilized metal-ions and a chemisorbed outer shell of a hydrophilic non crosslinked polymer and a method to produce such a product. It is possible to convert soluble polysaccharides to metal-binding glyco-conjugates. These glyco-conjugates include a plurality of metal chelating groups for each polymer molecule and, in addition, an excess of hydroxyl groups (or aldehyde functions etc.) which can be utilized for coupling biologically active components. The glyco-conjugate as well as similar conjugates of other polymers can be fixed to particles incorporating metal ions at their surfaces, e.g. magnetite, metal-ion derivated silica-gel particles etc.. In principle, it is possible to synthesize highly permeable lattices which permit adsorption of biological material from molecules to cells. Polymer coated metal particles and metal-oxide particles open new avenues for the separation, isolation and immobilization of biological macro-molecules and particles (virus, cells, etc.) both in the laboratory and in the workshop - techniques which compete on equal terms with and in certain instances are superior to chromatographic methods, or which can be used in instances where chromatography cannot be used at all. In respect of metal nuclei or cores in general, highly effective fluidized bed processes may be applied. When the particles are magnetic, these particles can be separated from solution and non-magnetic solids by placing them in a magnetic field. There are many methods by which the surfaces of metal can be coated with a polymer. The normal intention is to protect the surface of the metal with a plastics layer. In certain cases it may be desirable instead to affix solely an extremely thin layer of molecular thickness, for example the bonding of heparin to inner vessels and tubes in apparatus for through-pumping blood in surgical operations, so as to prevent coagulation. The invention is characterized in that the polymer is substituted with metal binding groups, chelaters, having at least three coordinating atoms, nitrogen, oxygen or thiolic sulfur, preferably as amine or carboxylated amine groups with the positions of the coordinating atoms such that each chelater is potentially able to form two or more adjacent 5- or 6-membered ring systems with a bridging metal ion. The method of fixing an organic polymer to a solid phase with immobilized metal ions at the phase boundary according to the invention is characterized by contacting said solid phase with an aqueous solution of the polymer substituted with metal binding groups, chelaters, having at least three coordinating atoms, nitrogen, oxygen or thiolic sulfur, preferably as amine or carboxylated amine groups with the positions of the coordinating atoms such that each chelater is potentially able to form two or more adjacent 5- or 6-membered ring systems with a bridging metal ion. Gel particles which have a heavy inner, impermeable and chemically resistant core have interesting fields of use within bio-technology. Chromatographic processes can often instead be conducted in beds at very high flow rates. This is true in those cases where there is unilateral material distribution between an immovable phase (fixed) and a movable phase. This is practically always the case in the biospecific adsorption of proteins, nucleic acids and polysaccharides, and also in a wealth of other adsorption processes for the aforesaid biopolymers. The adsorption process is greatly restricted by diffusion at high flow rates in the gel-particle beds. It is therefore important that the diffusion path is decreased, and consequently from this aspect the layer or coating should be as thin as possible. The bed must be given a very high specific surface area, in order to compensate for the low adsorption capacity. Both of these requirements can be satisfied by constructing the bed from extremely small, heavy particles with a diameter below 1mm embraced by a molecular polymeric layer or coating having bound thereto those centres which produce the adsorption. Our product according to the invention provides a solution to the problem and is based on a technique of fixing polymers, preferably water-soluble polymers, to surfaces which incorporate immobilized metal ions, particularly heavy metal ions belonging to the first series of transition elements, preferably iron, cobalt, nickel and copper, but also aluminium, zinc, cadmium, silver, gold and platinum metals. In principle, of course, other heavy metals, for example rare earth metals, aluminium, tallium, zirconium, thorium and uranium can also be surface treated with a molecular polymeric coating according to the invention. The main requirement is, after all, that insoluble metal complexes can be formed and consequently all insoluble metal compounds having superficial metal ions, such as oxides and sulphides, can be surface coated with polymers in accordance with the method of the invention. The manufacture of gel particles which incorporate magnetic cores has been known to the art since the 1950:s, when magnetite was embedded in ion exchangers. Polymer particles containing magnetite have also been used to increase the flow through filter cakes. The first magnetic gel particles for immobilizing proteins were described in 1973, and since then a number of articles and reviews have been published on the subject. Thus the USP No. 4 452 773 describes colloidal sized particles composed of iron oxide, coated with a polysaccharide or derivate thereof having pendant functional groups. These particles can be covalently bonded to antibodies, enzyme and other biological molecules. The particles are prepared by alcaline coprecipitation of FeCl₃, FeCl₂ and a polymer, preferably dextran. It is obvious that the invention described by us can be used for the same purpose. Example 1 illustrates how the invention can be applied to purify neuraminic acid-specific pilae from Coli-cells. Since pilae are extremely long and narrow molecules having a molecular weight in the order of 10⁶ Daltons, chromatographic gel material is not suited for cleaning purposes. Particles formed in accordance with the invention, however, present a readily penetrated polymer layer, which enables very high adsorption capacities to be achieved with both large and small molecules. Those manufacturing methods applied hitherto are based on the inclusion of a magnetic core in a gel-forming polymer, e.g. a polysaccharide such as dextran or agarose, or the polymerization of a suitable monomer, e.g. acrolein, in which magnetic particles are suspended and subsequently breaking-up the resultant gel composite. It is difficult, if not to say impossible, to obtain a uniform gel layer of given thickness by means of such methods. It must be admitted, however, that the polymerization of acrolein is a very neat solution to the problem, particularly since the polymer is "auto-activated" and can therefore be used immediately to couple proteins. On the other hand, the resultant polymer is quite hydrophobic and, above all, has limited permeability. We promote a novel technique for coating metal surfaces with a hydrophilic polymer such as a polyhydric alcohol, a polyamine, a polyamide, a polyether or a polyester, which can be expected to acquire a very wide usage. In our application of this "hydrophilizing technique" for separation and extracting purposes, we coat the metal surface with a non-cross-linked polymer. This means that practically the whole polymer is available to all molecules and particles, e.g. virus and cells. In this way there is obtained a macro-environment which is suitable for adsorption and which exhibits a very large adsorption surface area in relation to the surface area of the core particles. The method presumes that the surfaces of the metals (or metal compounds) are coated with metal ions, as are in fact all non-precious or base metals. The hydrophilized particle can be made heavy and also magnetic, by appropriate selection of the metal or metal compound. The primary layer or coating can be formed by "steeping" the particles in a polymeric solution and thereafter cross-linking the polymer. It is true that a thicker coating will be obtained, but it can still be highly permeable and presents a "frayed" outer surface. "Steeping" will also provide a more uniform polymer layer than those methods used hitherto for producing magnetic particles for adsorbing cells and molecules of biological interest. A further advantage lies in the possibility of building-up the layer or coating from different polymers. The product according to the invention can be obtained by reacting the superficial metal-ions in the solid phase - hereinafter referred to as M - with a group which binds strongly to M . L may suitably be metal-ion chelating or sulphide-forming sulphur, i.e. a thiol group. If L is bound to a polymer, there is immediately obtained therefrom a surface-coating in accordance with the invention. The product according to this version of the invention can thus be symbolized M-L-P. According to another version of the invention the polymer P* is coupled to M in another way, namely over a link or bridge -R-Y-located between L and P. In this case a substance L-R-X is allowed to react with a polymeric derivative Z-P, the substituents X and Z forming the bridge component Y by addition or condensation optionally followed by reduction. R or P can be a polyamide s.a. a polyacrylamide, a protein or a peptide, or a polyester s.a. an oligonucleotide or a polynucleotide Illustrations of such bridge forming reactions are: 2-aminomethyl-8-hydroxy quinoline one example of a substance of type L-R-x where L is and X is NH₂ and R represents remaining components of the molecule. It will thus be seen that when synthesizing the product, X and Z may be allowed to change places among the two reactants LRX and Z-P. The direction of the atoms in a chain will then be reversed. This is clear from the following example: LRX is and Z-P is carboxy-methylated agarose, symbolized as HOOC-CH₂- (agarose) to indicate the presence of reactive hydroxyl groups. The product then obtains the symbolic formula: If we depart instead from amino agarose and β -dicarboxymethyl propionic acid the following product is obtained: The bond Y is thus in the one case and the "retro- grade" sequence in the other. Particular mention must be made to two extremely useful reactions for producing the product according to the invention, namely the respective reactions of oxirane and divinyl sulphones with Z-hydroxyl-, amino(NH₂; NHR where R is alkyl or aryl) and thiol groups. LRX can then be illustrated symbolically as and L-R-SO₂-CH=CH₂ respectively when Z in ZP is OH, NH₂, NHR, or -SH. If X is OH, NH₂, NHR or -SH, coupling to the polymer can be effected when the polymer contains oxirane or vinyl-sulphonyl groups. The preparation of LRX as a pure chemical substance may be complicated. In this case there can be used in the preparation of a product according to the invention a mixture of components of which each fulfils the conditions for LRX. As an example, we can depart from tetraethylene pentamine. This substance forms strong complexes with Cu(II). It contains an excess of NH₂-groups and consequently fulfils the conditions for LRX when M is copper. Since the complexes formed with Fe(III) are very weak, the conditions for iron are not fulfilled. In this case the tetraethylene pentamine can be partially carboxy-methylated, therewith to obtain a mixture of differently substituted polyamine, e.g. and Several of the derivatives are able to form adsorption complexes with Fe(III) and other heavy metal ions and simultaneously have groups (-NH-or NH₂) capable of functioning as X for coupling to ZP or for activation with a group Z incorporating, for example, oxirane or a vinyl sulphonyl group. A practical application of the method for cases such as this is illustrated by Example 2 below. It may be difficult to produce the reactants LRX in a pure form. Pure reactants are not necessary in the synthesis of the product according to the invention. For example, if we depart from tetraethylene pentamine and carboxy-methylate this amine with insufficient monohaloacetic acid in alkaline solution (bromacetate for example), there is obtained a complicated mixture of derivatives, of which IV and V illustrate two examples. A further variant of the invention should be mentioned, namely where Y is also a metal complex. The following examples are given, in which LRX is and ZP is M = magnetite symbolized Fe Fe³ + . The product can therewith be described by the following formula: The preparation of the product according to the invention can be generally described in the following way: M₁ ... L₁---R---L₂...M₂...L₃P , where M₁ and M₂ may be the same or different metal-ions and where L₁, L₂ and L₃ may be the same or different metal-binding groups. Example 1 1.5 g magnetic particles (diameter approximately 0.4 µm) were incubated with 0.5 g iminodiacetate dextran (IDA-dextran) and shaken overnight in 0.5 M NaHCO₃-Na₂CO₃ buffer, pH 11. The IDA-dextran was herewith complex-bound to the metal particles. Subsequent to washing with distilled water followed by the above buffer, dextrans were activated with 1 % divinyl sulphone (DVS) dissolved in the buffer. After 1 h.40 mins, excess DVS was washed away with the buffer. The particles were then slurried in 10 ml of a buffer solution according to the above, containing 10 % dextran T 500 and allowed to stand at room temperature overnight. More dextran was herewith coupled to the surfaces of the particles. The procedure was repeated after washing the particles, although this time with a 5%-DVS. Finally the particles were activated with 2% DVS and divided into two equal portions. One portion was coupled with 0.15 g colominic acid in 1.5 ml buffer (pH 11). The other portion was coupled with 0.15 g transferrine in 1.5 ml bicarbonate buffer, pH 9.0. It was possible to elute 1.5 mg K99 pilae from E.coli for each gram of particles from the transferrine-substituted gel, and 0.65 mg/g from the colominic-acid particles. With regard to the colominic-acid particles, the result achieved was about 20 times more favourable than that achieved with colomine-substituted Sephadex G10. An estimation of the theoretical maximum adsorption capacity of a gel-coated magnetite preparation in which the magnetite has a specific surface of about 3 m²/g indicated that it should be possible to increase the capacity about a further ten times. The result achieved, however, is already highly satisfactory. Example 2. Magnetite particles coated with agar gel. Principle: To immobilize agarose with the aid of a novel technique based on metal-ion affinity 1. Chelaters + bifunctional activaters → monofunctional activated chelater derivative. 2. + Magnetite → particle surface coated monofunctionally activated chelater derivative → "active magnetite". 3. + Polymer → polymer covered magnetite (= end product). Routine : Partial carboxy-methylation of TEPA. 6 ml TEPA (tetraethylene pentamine) were added to 28 g bromacetic acid neutralized with NaOH + 100 ml 1 M Na₂CO₃/NaHCO₃, pH 11, and allowed to react for two hours at room temperature while shaken. 50 g powdered magnetite were added and the suspension shaken for two hours and washed with distilled water to a neutral pH by decantation. The product was finally washed with 0.1 M Na₂CO₃. Washing in distilled water (decantation) EDA-agar (corresponding to 1.25 g dry gel) was added and allowed to stand overnight while shaken. The pH was adjusted to 9.8 with Na₂CO₃. The product was washed in distilled water to a neutral pH.

Claims: Polymerconjugate for separation of biological molecules and particles consisting of a solid core with a surface containing immobilized metal ions and a surface coating consisting of a non-crosslinked polymer, the polymer being coordinated to the metal ions through chelating groups, each group with at least three identical or different coordinating atoms selected from the class amino-N, carboxyl or hydroxyl-O and thiol-S and with the coordinating atoms separated from each other by 2 or 3 carbon atoms. A product according to claim 1, characterized in that the chelater of the chemisorbed polymer contains one of the characteristic groups or A product according to claim 1 or 2, characterized in that the solid phase core is comprised of a metal or a metal oxide. A product according to claim 3, characterized in that the solid phase is in particle form and comprises particles having a diameter below 1 mm. A product according to claim 3 or 4, characterized in that the solid particles consist of magnetite. A product according to any one of the preceeding claims, characterized in that the chemisorbed polymer is a polyhydric alcohol, a polyamine, a polyamide, a polyether or a polyester. A product according to any one of the preceeding claims, characterized in that the polyhydric alcohol is a polysaccharide, for example dextran or agarose. A product according to any one of the preceeding claims, characterized in that the polyamide is a protein or a peptide. A product according to any one of the preceeding claims, characterized in that the polyester is an oligonucleotide or a polynucleotide. A product according to any one of the preceeding claims, characterized in that the hydrophilic polymer together with the chelater is incorporated in the surface of a particle, e.g. a virus or a cell or in a molecule aggregate. A method of fixing a water-soluble organic substance especially an organic polymer to a solid phase with immobilized metal ions at the phase boundary, characterized by contacting said solid phase with an aqueous solution of the polymer substituted with metal binding groups, chelaters, having at least three coordinating atoms, nitrogen, oxygen or thiolic sulfur, preferable as amine or carboxylated amine groups with the positions of the coordinating atoms such that each chelater is potentially able to form two or more adjacent 5- or 6-membered ring systems with a bridging metal ion. A method according to claim 11, characterized in that the hydrophilic polymer contains reactive groups such as vinylsulfone or epoxy groups in addition to the chelater groups. A method according to claim 11 or 12, characterized in that the metal-chelate forming groups are carboxymethylated amine.

Inventors: LINDAHL, MATS, PORATH, JERKER
Applicants: EXPLOATERINGS AB T.B.F.
Application year: 1985
Main classification: B01J20
IPC codes: C12N11, C07K1, C12N1, B01J20, G01N30, C12P1