MAGNETIC CARBOHYDRATE PARTICLES AS CARRIERS FOR AFFINITY SEPARATION PURPOSES, E.G. CELL SEPARATION

This invention relates to magnetically responsive cr stalline-carboh drate spheres or particles and to their use together with bioadsorptive materials. This includes coupling, adsorption and/or entrapment of bioadsorptive materials to said spheres or particles in connection with the use of interactions of these bioadsorptive materials with biological substances and cells. Preferably, in this invention use is made of magnetic nano-spheres .

By "magnetic nano-sphere" is meant a sphere or a particle the average diameter of which is 1-999 n and which is responsive to a magnetic field.

The advantage of using nano-spheres instead of micro-spheres is obvious from a calculation of the ratio of available surface area to volume unit of packed spheres :

A tenfold reduction of the diameter of the sphere gives a tenfold increase of the number of surface units per volume units of packed spheres. When bioadsor tive materials are to be used for covalent coupling or ad- sorption onto the surface of the spheres, the effort should be to use nano-spheres since the amount of bio¬ adsorptive materials per volume units of packed spheres is maximized. Furthermore, a nano-spnere is desirable in the following examples of separation, since the magnetic force to which for example a cell is exposed is considerably greater than in a case of a magnetic micro-sphere. This is due to the fact that a larger number of interactions is possible between nano-spheres and a cell compared to micro-spheres .

Examples of interactions which can be used in connec¬ tion with the present magnetic nano-spheres are as foll¬ ows :

1. protein-pro ein 2. protein-carboh drate

3. protein-lipid

4. protein-hapte , hormone or other low molecular weight substances, and combinations of the above interactions . With the use of said interactions there exists the possibility of separating heterogeneous biological mater¬ ial .

Examples of interactions of the above-mentioned type can be: cell separation, wherein a combination of inter¬ actions is included, inαnocheaical analysis, preferably where a pure interaction of any of the types 1—4 is involved depending on the purpose of the analysis, enzyme reaction, preferably when a protein-low molecular weight substance interacts, but also the types 1-3 are applicable, and, affinity purifying, when different types of inter¬ actions can be used depending on the substance to be purified.

Different types of magnetic spheres or magnetic particles are known since long in the literature for use such as in the above-mentioned interactions. A survey of magnetic spheres as carriers for bioadsorptive material is. published in Enz. Microb. Technol. Vol. 2, 1980 by Hailing & Dunhill. For example, the desired characteristics which a magnetic sphere or particle should have are mentioned therein: 1. The magnetic material shall be resistant to corrosion in aqueous surroundings.

2 - The polymer as such shall be chemically inert.

3. Bioadsorptive materials shall be able to couple or adsorb in a stable way to the surface of the micro- sphere . 4. The sphere shall, most preferably, be less than l μm.

5. The sphere shall contain enough magnetic material.

6. The spheres must not aggregate irreversibly at re¬ peated treatment in a magnetic field.

A number of different magnetic materials are de- scribed and commercially available, such as Fe- _j O^, Ni-NiO, Ni-TiO^, Mn-Zn ferrite, Ni etc. Generally, magnetite (FegO^) is used, since it is readily available and is not oxidized in aqueous surroundings.

Useful polymer materials are for example acrylic derivative, agarose, proteins, copolymers of acryl and agarose, starch and ^' cellulose. Among these neither starch nor proteins meet with the requirement of being chemically inert or resistant to degrading.

Known methods for producing magnetic micro-spheres are in general emulsion polymerizations with simultaneous cross-linking of the polymer to obtain stable micro- spheres. In one case the stabilization is obtained through heat-denaturation of protein (U.S. Patent 4 230 685). As polymer albumin (J.Pharm.Sci(1981) 70, 387-389) or dif- ferent acrylic derivatives (Nature (1977) 268, 437-438 or Science (1978) 200, 1074-1076) are used. The magnetic material is Various methods for coupling, ad¬ sorption or entrapment of the bioadsorptive material to/in the polymer are described in the literature and reference is made to Methods in Enzymology, vol. 44 (1976) (K. Mosbach) .

A method for producing starch micro-spheres in which the magnetic material is entrapped (FEBS Lett. (1979) 102, 112-116) has been described. However, this known technique is not applicable in the present invention, since the

obtained magnetic spheres are too large (2-3 microns) and are oxidized very quickly in aqueous surroundings due to the fact that the magnetic material is pure iron.

This problem can be avoided in a simple way according to the present invention, whereby magnetically responsive carbohydrate nano-spheres being resistant to corrosion can be produced.

DESCRIPTION OF INVENTION The invention relates to preparation and use of magnetically responsive spheres having an average diameter of 50-900. n and being built up of a polymer matrix in which magnetic particles having a diameter of 10—20 nm have been included. According to the present invention it is possible to prepare magnetically responsive carbohydrate spheres having a diameter of 50-900 nm. Thereby is obtained a large surface area per volume unit of packed spheres such that the possibilities of coupling of biological sub- stances is essentially higher than with other types of carbohydrate spheres (e.g Sephadex ^R or Sepharose ) . Due to the fact that the biological material is coupled to the surface of the spheres a more rapid reaction is also obtained since the diffusion barriers, occurring in a 200 micron sphere, are completely eliminated.

The matrix material in the preparation of the mag¬ netic nano-spheres according to the invention is a carbo¬ hydrate polymer. According to the present invention- use is made of the following carbohydrate polymers, dextran, pullulan, glycogen, starch, cellulose, agarose, alginate, chitosan or carrageenan. Dextran, cellulose and agarose are preferred and are used mainly since they are bio¬ chemically well characterized and inert in most biological systems. There is also the possibility of modifying carbohydrate polymers with for example acetyl, hydroxy-

propyl , propionyl, hydroxye h 1-hydroxypropanoyl , dif¬ ferent derivatives of acrylic acid or similar subεti- tuents .

The present process relates to preparation of mag- netic spheres having an average diameter of 50-900 n . The process is characterized in that the carbohydrate polymer is dissolved in a solvent having a high dielectric con¬ stant to a concentration normally within the range of 0.1-150% (w/v). Useful examples of such solvents are inter alia dimethyIformamide , ethylene glycol, sulfolane, dimethyl • sulfoxide , propylene carbonate, water and form- amide or mixture of these. The magnetic material is suspended in the dissolved carbohydrate polymer. The magnetic material consists of magnetite having a size of 10-20 nm. A method for preparation of such small particles is known and these are commercially available via Ferrofluid Corp., U.S.A. Other useful magnetic ma¬ terials include particles or colloids ha ing a main content of aluminum, nickel, cobalt, copper, silver, manganese or platina.

The so obtained suspension of magnetic material in the dissolved carbohydrate polymer is emulsified in an emulsion system consisting of said magnet suspension and an emulsion medium consisting of a liquid which is im- miscible with said magnet suspension. A further charac¬ teristic of the liquid is that it participates in the formation of. small drops of magnet suspension in the emulsion medium.

Examples of useful emulsion media may be vegetabilic oils, preferably rape-oil or maize-oil, and other useful hydrophobic emulsion media include paraffin oils or silicon oils .

Another type of emulsion medium comprises organic solvents in which one or more emulsifiers have been dissolved. Useful such organic solvents comprise inter

alia xylene, toluene, ethyl benzene, diethyle benzene, propyl benzene, ethylene chloride and other similar solvents and mixtures thereof.

In order to emulsify the emulsion sonicates or a high-pressure homogenizer are used. The obtained emulsion, in which the carbohydrate magnetite suspension is emul¬ sified in form of drops, is stabilized by transferring same into a liquid the characterisric of which is that it can crystallize the carbohydrate polymer, whereby the magnetic material is enclosed.

Useful such liquids are ethanol, methanol and ace¬ tone, preferably acetone. After crystallization the nano-spheres are further washed with acetone. Drying can be realized in a simple way through rotatory evaporation or air-drying.

The present invention furthermore relates to co- valently coupling of bioadsorptive materials onto the surface of the magnetic nano-sphere.

Example 1

0.45 g dextran, having a molecular weight of 40,000 (Pharmacia AB, Uppsala), was dissolved through warming in a mixture consisting of 700 ul H _j O and 300 ul Ferrofluid (EMG 806,200 Gauss, Ferrofluid Co., U.S.A.). The solution was allowed to cool to room temperature, whereafter it was transferred into a 100 ml beaker containing 25 ml cold (+4°C) vegetabilic oil. The mixture was emulsified with ultrasonic (Ultrasonic 350 G) for 1 minute, whereafter the obtained emulsion was poured into 200 ml acetone, ^' in which the emulsifier Tween 80 has been dissolved to a con¬ centration of 0.1% (w/v). While thoroughly pouring the emulsion into acetone-Tween 80 it was stirred at about 1000 rp . The crystallized magnetic nano-spheres were washed 4 times with said acetone solution, whereafter they were air-dried.

Example 2

The same procedure as in example 1 was followed, except for the use of 150 mg magnetite particles (EMG 1111) instead of 300 _μ l Ferrofluid.

Example 3

The same procedure as in example 1 was followed, except for the use of xylene with the emulsifiers Gafac PE-520 and Tween 80, both with a concentration of 5% (w/v), instead of the vegetabilic oil.

Example 4

The same procedure as in example 1 was followed, except for the fact that the dextran was crystallized in ethanol with addition of 1 g Tween 80 per litre ethanol instead of acetone-Tween 80.

Example 5

The same procedure as in example 1 was followed, except for the fact that 0,3 g starch was dissolved in 700 μl formamide and 300 ml Ferrofluid (EMG 2111) instead of dextran in H2O.

Example 6 The same procedure as in example 1 was followed, except for the fact that dextran having a molecular weight of 500,000 instead of 40,000 was used.

Example 7 The same procedure as in example 1 was followed, except for the fact that 0.8 g 2% agarose or carrageenan was mixed with 300 μl Ferrofluid at a temperature of +50°C instead of dissolving the dextran in water and Ferrofluid.

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Example 8

The same procedure as in example 1 was followed, except for the fact that 0.8 g 2% alginate instead of dextran and water was mixed with 300 μl Ferrofluid and emulsified in the vegetabilic oil containing 0.1% (w/v) of the emulsifier Gafac RM-410 instead of pure oil.

Example 9

The same procedure as in example 1 was followed, except for the fact that 0.8 g 1% chitoεan, dissolved at pH 5, was mixed with 300 μl Ferrofluid instead of dextran, water and Ferrofluid.

Example 10 The same procedure as in example 1 was followed, except for the fact that 3 g α-cellulose was dissolved in 150 g N-ethylpyridine chloride and 75 g dimethylformamide, and 0.8 g of this solution was mixed with 300 ul Ferro¬ fluid instead of dextran, water and Ferrofluid.

Example 11

25 mg of the nano-spheres obtained in example 1, activated with tresylchloride , was suspended in 1 ml 10 mM phosphate buffer, pH 7.2, containing 0.9% NaCl , where- after 20 mg labled (fluoroscein) bovine serum albumin (BSA*) was added. The protein was coupled during 15 minutes at room temperature and was compared with the case where the nano-spheres were dissolved directly in phos¬ phate buffer containing BSA*. Non-coupled BSA* was washed (3 times) off with phosphate buffer; the nano-spheres were kept stationary by means of a small hand magnet. After washing the result was checked by means of a Niko fluor¬ escence microscope and it was established that all nano- spheres were fluorescing strongly and no difference between the two ways of coupling was detected.

Example 12

The magnetic nano-spheres were activated with bromo- cyanide (60 mg BrCN/gr wet gel) at pH 10.75 during 11-12 minutes. 500 μl anti-alphafetoprotein-antibodies were coupled to the gel over night at +4°C.

This magnetic antibody-gel was used to measure the serum level on alphafetoprotein (AFP) by means of a so-called sandwich-ELISA. Thus, after mixing the nano- spheres with test serum and incubation for 2 hours, the spheres were washed by applying small hand magnets outside the test tubes containing serum sample and spheres, whereafter the wash buffer was discarded. The washing procedure took 5-10 minutes. Thereafter anti-AFP con¬ jugated to glucose oxidase was added. After 2 hours of incubation and following washing the enzyme substrate was added and the colour reaction was followed by a photometer after 30 minutes. This sandwich enzyme immunoassay gives a standard curve in which the absorbance is proportional to the amount of AFP in the sample. The assay time was about 5 hours, the sensitivity 10-50 ng AFP/ml sample, and the test capacity was about 100 samples/day.

Example 13

5 mg magnetic nano-spheres according to example 1, activated with tresylchloride , were suspended in 300 μl phosphate buffer, pH 7.2, containing 0.9% NaCl and 25 μl goat anti-mouse IgG (49.8 mg/ml). The suspension was incubated for 30 minutes at room temperature and washed with phosphate buffer 3 times by means of a magnet which held the spheres in place. After washing the spheres were resuspended in 0.5 ml phosphate buffer, pH 7.2, containing 0.5% NaCl. To this were added 10 human peripherial lymphocytes, pre-incubated with monoclonal antibodies of anti-0KT4 specificity (0KT4 is a marker of a T-cell).

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The cell and sphere suspension was incubated for 60 minutes at 37°C, whereafter the test tube was exposed to a magnetic field. Cells coated with the magnetic nano- spheres were held stationary by the magnet, and the cells were washed twice with pnosphate buffer. The separated cell population was about 40% of added lymphocytes and no (<1%) contamination of non-0KT4 positive cells could be detected.

Example 14

To 5 mg magnetic nano-spheres, activated with tresyl- chloride, were coupled a carbon hydrate-binding protein. This lectine, phytohemaglutine (PHA) , binds to hydrate residues on cell surfaces or free in solution. PHA posi- tive magnetic spheres (0.5 mg/g wet gel) were incubated with red blood corpuscles (RBC) from rabbit for 60 minutes at 25°C. The suspension was pumped in a tube exposed to a magnetic field of about 3000 gauss, whereby RBC was retained where the tube passed the magnetic field. This shows that even relatively weak interactions of the type lectine-carbon hydrate, association constants are about 10 -10 M~ , can be used for cell separation. When mag¬ netic nano-spheres were used very little contamination of non-bonded cells is obtained in the outflow for reasons already discussed.