Polymer coated particles having immobilized metal ions on the surfaces thereof

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 adsorbed on the solid phase. The polymer layer may constitute a polyhydric alcohol, a polyamine, or a polyamide, for example a polysaccharide, such as agarose, a protein or a peptide, a polyacryl amide, etc. and the reactive group may be a vinylsulphone 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.

A novel method of coating the surface of a metal or insoluble metal 
compound with a monomolecular polymeric layer is IMA-technique 
(Immobilized Metal Ion Affinity) 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. 
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 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. 
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 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. 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 10.sup.6 
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. 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, 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. 
Illustrations of such bridge forming reactions are: 
2-aminomethyl-8-hydroxy quinoline 
##STR1## 
one example of a substance of type L-R-X where L is 
##STR2## 
and X is NH.sub.2 and R represents remaining components of the molecule. 
The reaction with M produces an adsorption complex (adsorbate) of a 
chelate nature, which in turn is able to react with a reactive polymer, 
e.g. an aldehyde starch, ZP with Z=--CHO. If the reaction product is 
reduced, for example, with sodium borhydride, there is obtained a stable 
coupling of the starch derivative to the 8-hydroxy quinoline, which is in 
turn firmly bonded to the metal: 
##STR3## 
Reactions can also be used in another sequence to produce the product 
according to I. If LRX is instead based on 8-hydroxy quinoline-2-aldehyde, 
the polymer may include Z=NH.sub.2, e.g. an amined aldehyde starch. The 
product I is obtained subsequent to adsorption of the aldehyde on the 
metal-ion followed by reduction. 
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: 
##STR4## 
and Z-P is carboxy-methylated agarose, symbolized as HOOC--CH.sub.2 -- 
(agarose) to indicate the presence of reactive hydroxyl groups. The 
product then obtains the symbolic formula: If we depart instead from 
amino agarose and .beta.-dicarboxymethyl propionic acid the following 
product is obtained: 
##STR5## 
The bond Y is thus --NH--CO-- in the one case and the "retro-grade" 
sequence --CO--NH-- 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(NH.sub.2 ; NHR where N is alkyl or aryl) and thiol groups. LRX can 
then be illustrated symbolically as 
##STR6## 
and L--R--SO.sub.2 --CH.dbd.CH.sub.2 respectively when Z in ZP is OH, 
NH.sub.2, NHR, or --SH. If X is OH, NH.sub.2, 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 
NH.sub.2 --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. 
##STR7## 
Several of the derivatives are able to form adsorption complexes with 
Fe(III) and other heavy metal ions and simultaneously have groups (--NH-- 
or NH.sub.2) 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 
##STR8## 
M=magnetite symbolized FeFe.sup.3+. 
The product can therewith be described by the following formula: 
##STR9## 
The preparation of the product according to the invention can be generally 
described in the following way: 
EQU M.sub.1 . . . L.sub.1 --R--L.sub.2 . . . M.sub.2 . . . L.sub.3 P 
where M.sub.1 and M.sub.2 may be the same or different metal-ions and where 
L.sub.1, L.sub.2 and L.sub.3 may be the same or different metal-binding 
groups.

EXAMPLE 1 
1.5 g magnetic particles (diameter approximately 0.4 .mu.m) were incubated 
with 0.5 g iminodiacetate dextran (IDA-dextran) and shaken overnight in 
0.5M NaHCO.sub.3 --Na.sub.2 CO.sub.3 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 m.sup.2 /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.fwdarw.monofunctional activated 
chelater derivative. 
2. +Magnetite.fwdarw.particle surface coated monofunctionally activated 
chelater derivative.fwdarw."active magnetite". 
3. +Polymer.fwdarw.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 1M Na.sub.2 CO.sub.3 /NaHCO.sub.3, 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 0.1M Na.sub.2 CO.sub.3. 
##STR10## 
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 Na.sub.2 CO.sub.3. 
The product was washed in distilled water to a neutral pH.