SE451715B - POROST MATERIAL WITH A BIOLOGICALLY ACTIVE SUBSTANCE IMMOBILIZED ON THE SURFACE AND PREPARATION OF IT - Google Patents

Description

The invention also relates to the method for producing material according to the invention.

By "sorptive inorganic material" is meant herein that the inorganic material is either sorptive itself or has been treated to become sorptive and able to sorb molecules. Sorptive processes include absorption and adsorption, which includes physical adsorption and chemisorption, and combinations thereof. .

By the term "substantially insoluble" is meant herein that the inorganic material is substantially insoluble in substances with which it comes into contact during the preparation of the porous material according to the invention and in the use of such porous materials according to their intended use.

It is generally advantageous to at least partially dry the material after the additive has been insolubilized, in order to facilitate handling and / or storage. Drying can e.g. in air or methanol.

In preparing the present material, the slurry is converted into droplets which are contacted with a reactant which can precipitate the insoluble form of the additive to give substantially spherical discrete particles of an intermediate, so that substantially spherical discrete porous particles is formed after removal of the precipitated insoluble additive. The intermediate is conveniently separated from the precipitating reactant before being treated to remove the precipitated form of the additive.

The insoluble form of the additive is conveniently removed by heating the intermediate to a temperature at which the insolubilized additive is "burned off" to give a porous material, i.e. the insolubilized additive acts as a volatile additive.

In addition to the additive, another porosity-forming volatile material can be introduced into the intermediate product and then used to effect porosity in the porous material, thereby serving to increase or modify the porosity produced upon removal of the insolubilized additive. 451,715 British Patent Specification 1,421,531 describes i.a. a method * for producing discrete porous particles with internally coherent pores for selective retention of predetermined macromolecules from a liquid medium. The method of the invention was used to prepare discrete porous particles and immobilize such macromolecules. In this case, the porous material should have such a pore structure that allows the molecules to penetrate into the material and be sorbed by the sorptive inorganic material. British Pat. No. 1,421,531 further elucidates the pore size and its relation to the size of the molecules which can be sorbed or excluded from sorption.

The pore size of the porous material and thus the size of the molecules that are excluded or allowed to penetrate into the particles can be varied by varying the amount of additive or volatile agent, or by introducing suitable additives or volatiles in the manufacturing step.

In some cases, inorganic ions may be introduced into the intermediate, e.g. by interaction with the additive so that a complex is formed, and give rise to impurities in the porous material after heating. If these impurities are soluble, they can be removed by treating the porous material with a suitable solvent.

Examples of additives which can be converted into an insoluble form and which can be used for the preparation of a material according to the invention are high molecular weight materials such as alginic acid, alginates, pectin, polygalacturonic acid and proteins. These additives are preferred for economic reasons, but other expensive additives which can be converted to insoluble form may be used, e.g. carboxyethylcellulose derivatives, rubber, which give insoluble products with acids or cationic solutions, derivatives of polyacrylic acid, polymers of styrene maleic anhydride and nucleic acids.

By "insoluble form" with respect to the additive is meant that it is substantially insoluble in substances used during the formation of the intermediate, for example reactants used in the precipitation and substances used in the further treatment of the intermediate, eg desiccants. Examples of additional porosity-forming volatile materials that can be incorporated into the intermediate, in addition to the additives that can be converted to insoluble form, are hemoglobin and soluble starch and powdered insoluble polymers. casein, gelatin and vegetable proteins as well as "volatile additives" described in the above-mentioned British patent specification, eg ammonium carbonate, polyvinyl alcohol, dextran, ovalbumin, ox-serum albumin and In the selection of the additional porosity-forming volatile material, it is appropriate to should choose a substance that does not diffuse or leach easily s from the slurry during the conversion of the additive to insoluble form. It should also not give rise to premature precipitation, ie before the drip formation. For example, when an alginate is used as an additive, certain casein preparations containing calcium ions as a contaminant should not be used, as these ions cause premature precipitation of calcium alginate.

It is assumed that the additive and the porosity-forming volatile material contribute to the formation of the interconnected pores, ie a porous network, in the porous material.

Examples of inorganic materials which can be converted into porous materials according to the invention are titanium oxide, calcium phosphate, certain natural products such as bentonite and celite, and other inorganic materials disclosed in British Patent Specification 1,421,531, i.e. alumina, barium sulphate, zinc oxide and calcium sulfate.

Suitably discrete porous particles according to the invention are prepared by a finely divided, substantially insoluble, sorptive inorganic material and an aqueous solution of a soluble alginate, e.g. sodium alginate, is mixed into a slurry which is converted into droplets which are contacted with a reactant, e.g. aqueous calcium chloride solution, which can precipitate the soluble alginate in insoluble form to thereby form particles of an intermediate containing particles of inorganic material bonded by the precipitated alginate, and heating the intermediate particles to remove at least a portion of the alginate to thereby form discrete porous particles, after which a now biologically active substance is brought into contact with the particles.

Conveniently, the discrete porous particles according to the invention can also be prepared by a finely divided, substantially insoluble, sorptive inorganic material and an aqueous solution of a soluble alginate, e.g. ammonium alginate, is mixed into a slurry which is converted into droplets which are contacted with an acidic reactant to precipitate alginic acid to thereby form intermediate particles containing particles of the inorganic material bonded by the precipitated alginic acid, and heating the intermediate particles to remove at least a portion of the alginic acid to form discrete porous particles, after which the particles are brought into contact with a biologically active substance.

In both of the foregoing embodiments, the slurry may optionally contain an additional porosity-forming volatile material to provide increased or modified porosity in the discrete porous particles.

Discrete porous particles that can selectively retain macromolecules in a liquid medium have been prepared from titanium oxide, celity bentonite and calcium sulfate with a particle diameter of 200-500 μm with pore sizes sufficient to let in macromolecules which are then sorbed. The particles can be produced in sizes suitable for special uses and can, if desired, be produced in sizes outside the above range. Depending on the use, the particles can be produced in sizes of e.g. 50} nn - 5 mm diameter.

An example of a macromolecule that is selectively retained on discrete porous particles of the invention is hemoglobin, which is selectively retained from an aqueous solution containing hemoglobin and ox-serum albumin. Examples of other macromolecules that can be selectively retained are given in British Patent Specification 1,421,531.

It has been found that a number of factors affect the size of the discrete porous particles of the invention. Thus, the size depends on the amount of inorganic material in the slurry, the droplet size of the slurry before the conversion step, for example the precipitation step, and the degree of porosity. The droplet size depends on the viscosity of the slurry and when a nozzle is used to make the droplets on the nozzle orifice diameter, type of nozzle e.g. needle, vortex or atomizing nozzle, and the rate at which the slurry is ejected from the orifice.

These factors can be varied to produce particles of the desired size.

On the porous material of the present invention, as biologically active substances, e.g. enzymes be immobilized, e.g. according to the invention set forth in British Patent Application No. 28 212/74, corresponding to the German publication 25 27 884.

Thus, amyloglucosidase has been immobilized on discrete porous particles of TiO2 and celite.

The invention is described in more detail by the following examples, in which examples 1-8 illustrate the process the first part of the process, namely the preparation of porous particles and Example 9 illustrates the last part of the process.

Example 1 200 g of TiO 2 were slurried in 1 liter of a 1% aqueous solution of sodium alginate (Welgum Alginate Industries Ltd.) by ball milling for 5 hours to give a slurry of suitable viscosity to form droplets. The slurry was added dropwise through a pipette nozzle (1 mm diameter) to a solution of 0.1 M calcium chloride to form discrete particles of an intermediate containing TiO 2 particles bonded by precipitated calcium alginate. These particles were strong enough for handling and transferred to methanol, where they were allowed to dehydrate to 25% of the original volume. Thereafter, the particles were heated in an oven to 100 ° C for 1-2 hours and finally sintered at 90,000 to give discrete porous particles of titanium oxide of 500 microns in diameter.

Example 2 200 g of celite were slurried in 1 liter of a 2% aqueous solution of sodium alginate (Welgum Alginate Industries Ltd.) by grinding in a ball mill for 5 hours. In the same manner as in Example 1, the slurry was added dropwise to a 0.1 M calcium chloride solution to give discrete particles of an intermediate product consisting of particles of celite bonded by precipitated calcium alginate. The particles were dried 431 451 7.15 in air and then in an oven at 100 ° C and finally sintered. o. at 1150 DEG C. to give discrete porous particles of celite with a diameter of 500 .mu.m. Example 3 200 g of TiO2 were slurried in 1 liter of a 2% aqueous solution of ammonium alginate (Collatex A / RK EXTRA, Alginate Industries Ltd.) using a homogenizer for 20 minutes. The slurry was added dropwise through a 1 mm diameter pipette nozzle to 0.1 M HCl to form discrete particles of an intermediate containing TiO 2 particles bound with alginic acid. These were dried in air and heated to give discrete porous particles of titanium oxide.

Example 4 To a sample of the slurry prepared according to Example 1, 10% by weight of hemoglobin was added as an additional porosity-forming volatile material. The slurry was converted to droplets and treated as in Example 1 to give discrete porous particles.

Example 5 The procedure of Example 4 was repeated, except that 10% by weight of soluble starch was added as an additional porosity-forming material to give discrete porous particles.

Example 6 600 g of celite (eg Koch-Light Laboratories Ltd.) and 10 g of soluble starch (eg BDH Ltd.) were slurried in 6 liters of a 1% aqueous solution of sodium alginate by homogenization in a homogeneous - icing machine for 20 minutes; The resulting slurry was added dropwise to a 0.1 M aqueous calcium chloride solution to give discrete particles of an intermediate containing celite particles and starch bound with calcium alginate. The discrete particles were dried in methanol and then in air at 80 ° C and then heated to 1150 ° C to give discrete porous particles of celite with a diameter of 500 μm »Example 7 20 g TiO 2 and 5 g of the commercially available polymer resin Amberlite CG50 100-200 mesh as an additional porosity-forming material was slurried in 100 ml of a 1% aqueous solution of sodium alginate. The resulting slurry was added dropwise to a 0.1 M CaCl 2 solution to give discrete particles consisting of calcium phosphate bound by precipitated calcium alginate. It turned out that the particles could be dried in air or methanol and give particles strong enough for handling. The particles were heated to give discrete porous particles of calcium phosphate.

Example 8 20 g of calcium orthophosphate were stirred in 100 ml of 1% aqueous sodium alginate solution of the type used in Example 1, and without delay the resulting slurry was added to a 0.1 M CaCl 2 to give discrete particles consisting of calcium phosphate. bound by precipitated calcium alginate. It turned out that the particles could be dried in air or methanol and give particles strong enough for handling. The particles were heated to give discrete porous particles of calcium phosphate. Example 9 According to this example, a biologically active substance, in this case the enzyme papain, was immobilized on discrete porous particles.

Thus, 5 ml of discrete porous particles of celite prepared as in Example 6 were mixed thoroughly with 1.5 ml of Pasol on ice, where "Pasol" is a papain preparation in solution from Powell and Schofield.

A 4% solution of Tannia which is a synthetic polyphenol from e.g.

Harshaw Chemicals in 2: 1 aqueous acetone was prepared and the pH was adjusted to 7 with NaOH. To 2.25 ml of this solution was added 0.6 ml of formaldehyde. The resulting solution was added to the celite particles and Pasol, after which the whole was left on ice for 75 minutes. V I Kf 'The resulting celite particles on which the papain enzyme had been immobilized were washed in demineralized water and tested for enzyme activity using benzoyl-alginine ethyl ester as substrate and measured the esterase activity. The insoluble activity was 10% of the soluble activity. 7

Claims (4)

1,451,715 Claims Porous material with a biologically active substance immobilized on the surface, characterized in that it is prepared by a slurry consisting of particles of a finely divided, mainly insoluble, sorptive, inorganic material, an additive and a solvent for the additive, wherein the additive can be converted into insoluble form, is treated by converting the slurry into droplets, which are contacted with a reactant which precipitates the insoluble form of the additive, thereby forming an intermediate of the particles of the inorganic form. the material bonded by the insolubilized additive in the form of substantially spherical, discrete particles and that the intermediate is treated to remove at least a portion of the insolubilized additive to form the porous material in the form of substantially spherical, discrete particles, wherein the inorganic the material, the additive and the amount of additive selected accordingly in such a way that the porous material has an internally coherent pore structure, which is such that macromolecules can penetrate into the material, and that a biologically active substance is brought into contact with the material. Porous material according to claim 1, characterized in that the discrete porous particles have a diameter in the order of 50 μm - 5 mm. A porous material according to claim 2, characterized in that the discrete porous particles have a diameter of the order of 200 - 500 μm. A process for the preparation of a porous material according to claim 1 with a biologically active substance immobilized on its surface, characterized in that a slurry consisting of particles of a finely divided, substantially insoluble, sorptive, organic material, an additive and a solvent for the additive, wherein the additive can be converted into insoluble form, is treated by converting the slurry into droplets, which are brought into contact with a reactant which precipitates the insoluble form of the additive, thereby forming an intermediate 451 715 Product of the particles of the inorganic material bonded by the insolubilized additive in the form of substantially spherical, discrete particles and the intermediate being treated to remove at least a portion of the insolubilized additive to form the porous material in the form of substantially spherical, discrete particles, the inorganic material, the additive and men The additive is selected in such a way that the porous material has an internally coherent pore structure, which is such that macromolecules can penetrate into the material, and that the biologically active substance is brought into contact with the produced porous material for immobilization. on its surface.