DE3432143A1 - METHOD FOR ENCLOSURE A MATERIAL TO BE ENCLOSED - Google Patents

Description

3432H3

Damon Biotech, Inc. Needham Heights, MA o2 194, V. St. A,

Method of encapsulating an encased

Materials

The invention relates to a method for encapsulating a material to be enveloped in a semipernvable Membrane, in particular a method for encapsulation in a microcapsule of a material, including live cells that are sensitive to pH, temperature or ion concentration is. The encapsulation method according to the invention enables the formation of a semipermeable membrane without damage to the material to be wrapped. The invention also relates to a capsule in which an inner layer made of an aminated polymer ionically bonded to an outer layer made of an anionic polymer is.

Although a variety of methods for microencapsulating material to be encapsulated have been developed most of these procedures cannot be performed because of the harsh conditions required for them Encapsulation of material such as living cells

0192-DBH-419-K1-E

that is sensitive to pH, temperature or ion concentration. In the U.S. Patent 4,352,883 is believed to be the first method of successfully encapsulating living tissue or described cells in a semipermeable membrane. An intermediate capsule is created around the tissue or cells formed from a gellable material, preferably an anionic gum such as sodium alginate; a permanent, semipermeable membrane is produced by crosslinking the surface layers of the intermediate capsule. Typically, a mixture of the rubber and the material to be wrapped is used to make a Intermediate capsule treated with a gelling solution, preferably a calcium ion solution. The resulting intermediate capsule is used to manufacture a permanent membrane with a solution of a polycationic material implemented. The interior of the capsule can be re-liquefied by restoring conditions among which the anionic gum is liquid, e.g. B. by changing the ion concentration upon introduction of the capsules in a phosphate buffered saline solution. The reliquefaction of the interior of the capsule facilitates the transport of nutrients through the membrane and promotes cell growth. Since the conditions for encapsulation, such as temperature, ionic strength or pH range, do not necessarily have to be sharp, is also damage of the material to be enveloped or impairment of the viability of the cells is not absolutely necessary.

The object of the invention was therefore to provide a method for encapsulating living cells or other sensitive

available in semipermeable membranes to deliver. Also should be encased material because of its sensitivity to pH, the Ion concentration or charge or the temperature is difficult to encapsulate in known processes, in the invention Procedure to be encapsulated. Another object of the invention was to provide an improved capsule to provide with a semipermeable membrane.

These objects are achieved according to the invention.

The invention therefore relates to a method for encapsulating a material to be enveloped in a semipermeable membrane, which is characterized in that

A. the material to be enveloped in an aqueous medium in which a polysaccharide containing cations is dissolved, is suspended,

B. the suspension is formed into droplets containing the material to be enveloped,

C. The droplets are treated with a solution containing anions, whereby they become discrete, dimensionally stable Gel intermediate matrices, and

D. the intermediate matrices are treated with a polymer containing anions that react with the cations, wherein the surface layers of the intermediate matrices are crosslinked into capsules around the droplets.

3A32U3

In the method according to the invention, material to be enveloped, such as enzymes, antibodies, hormones or living cells, z. B. tissue cultures or genetically modified cells, encapsulated in a semipermeable membrane will. The material to be enveloped is suspended in an aqueous medium by containing a cation Polysaccharide is dissolved, preferably an aminated polysaccharide, such as a poly-D-glucosamine (chitosan). The droplets of the suspension gel when treated with a solution of a divalent or polyvalent anion, z. B. with phosphate, hydrogen phosphate or sulfate salts. A permanent membrane is created by networking the Surface layers of the intermediate matrix with a polymer that contains anions and preferably carboxyl groups, the react with the cations of the matrix. Poly-L-glutamic acid and poly-L-aspartic acid, either as such or in the form of its salts, are preferred anionic polymers. The inside of the capsule can be cleaned by treating the capsule with a solution of a low molecular weight polycation, z. B. spermadine, spermine or urea, can be made soluble again. The reliquefaction of the interior of the capsule promotes mass transport between the extracapsular fluid and the intracapsular space.

Live cells can be used in the method according to the invention encapsulate in a semi-permeable membrane. The cells are placed in an aqueous medium that increases viability of the cell is not affected and contains an aminated glucopolysaccharide. The droplets the suspension are treated with a gelling solution, creating a dimensionally stable, water-insoluble intermediate matrix arises. The gelling solution is an aqueous solution

solution of a divalent or polyvalent anion, e.g. B. a solution of phosphate, hydrogen phosphate or sulfate salts. The capsule is then coated with a polymer containing a variety of carboxyl groups, e.g. B. polyglutamic acid or polyaspartic acid, treated, whereby the surface layers the intermediate matrix are permanently networked. The interior of the capsule is practically free of precipitation Reaction liquefied again if you put the capsule in a solution of a low molecular weight polycation, for example Exposing spermadin, spermine or urea. Reliquefaction takes place by removing the polyvalent anions from the inner gel. Human or animal tissue cells from a tissue growth medium and genetically modified, procaryotic or eukaryotic Cells in a growth medium can in the invention Procedure to be encapsulated. The inventive method for encapsulation is so gentle that the encapsulated Cells are not damaged; the normal metabolic processes, including growth and reproduction, continue within the capsule.

The invention also relates to a capsule in which a membrane forms a closed intracapsular space surrounds. The membrane has an inner layer made of a polyaminated polymer and preferably an aminated one Polysaccharide, especially an aminated glucopolysaccharide, ionically attached to an outer layer made of a polyanionic polymer and preferably a polycarboxylated one Polymer, such as polyglutamic or polyaspartic acid, into a water-insoluble permanent capsule is networked. One can get into the intracapsular space

Embed cell, preferably a eukaryotic, bacterial or genetically modified cell; in this case the polyanionic and polyaminated polymers should be physiologically compatible with the cell.

The invention relates to a method of encapsulating a material to be enveloped, such as eukaryotic or prokaryotic, naturally occurring or genetically modified live cells or tissue cultures in a semipermeable membrane. The invention also relates to a new multilayer capsule.

By reaction of a polysaccharide containing cations or glucopolysaccharide with a polyvalent anion creates a gel or intermediate matrix the material to be enveloped or the living cell. The surface layers of the resulting intermediate matrix are with a polymer that contains anions and preferably carboxyl groups, into a permanent one to be enveloped Material encapsulating membrane ionically cross-linked. The inside of the capsule can be made by treating the capsule with be liquefied again with a solution of a low molecular weight polycation.

The material to be wrapped, e.g. B. enzymes, hormones, antibodies or live cells is in an aqueous Suspended medium which contains a gellable, cation-containing polymer. A preferred gellable Polymer is an aminated glucopolysaccharide, especially chitosan (poly-D-glucosamine). Chitosan is created by acid hydrolysis of chitin (poly-D-N-acetylglucosamine), which is the first structural substance of the invertebrate exoskeleton. Chitosan is a long-chain, aminated

Polymer that is physiologically compatible with most cells. It is only slightly soluble in water, it however, it can easily be dissolved in dilute acetic acid.

Chitosan stock solutions are made by dissolving the chitosan in 0.5 M (mol / l) acetic acid. The acetic acid is removed from the solution by repeated dialysis against phosphate buffered saline (PBS). The chitosan does not precipitate from this solution, if kept below 37 ⁰ C. The preferred stock solution, 1.4 % chitosan in PBS, was successfully stored ^{at 4 ° C. for a longer period of time.}

The chitosan prepared as described above is mixed with the material to be enveloped, forming a solution or a slurry which is then formed into droplets or other shapes. Although solutions with a final concentration of chitosan of 0.4 % (weight / volume) gel, the optimum gelation is between 0.8 and 1.2 l (weight / volume) chitosan in isotonic, 125 mM Na-HPO. Solution . The suspension of chitosan and enveloping material can be formed into droplets using any conventional droplet-forming device. Such a droplet-forming device is explained below.

A tube containing the suspension is equipped with a closure on which the droplet-forming device is appropriate. The device consists of a housing that has an air injection nozzle on top and a elongated frictional hollow body, which is inserted into the closure

fits, has. A 10 ml syringe that comes with a Step pump is connected and has a needle that extends the length of the housing, for example a Teflon-coated needle with an inner diameter of 25 mm is attached to the top of the housing. The interior of the housing is designed so that the tip of the needle is exposed to a constant stream of laminar air that acts as an air knife. In operation, the step pump pushes droplets of solution from the tip of the needle. Each drop is "cut off" by the air stream and falls approximately 2.5 cm into the gelling solution, preferably a disodium hydrogen phosphate solution, where it immediately gels through reaction with the negative ions. Of the The distance between the needle tip and the surface of the gelling solution is preferably large enough to allow the suspension to give the physically most favorable shape of chitosan and the material to be enveloped, d. H. one Spherical shape (maximum volume with a minimum surface). The air from the pipe flows through an opening in the lock. This process ensures "crosslinking" of the gel and the formation of a highly viscous, dimensionally stable, protective intermediate matrix that contains the suspended material to be enveloped and its medium contains. The intermediate matrices collect in the solution as a separate phase and can be removed by suction be separated.

As a polyvalent gelling solution, a 125 mM Na ^ Solution preferred; however, sodium phosphate and sodium sulfate monobasic solutions also give acceptable intermediate matrices. Sodium citrate also gels the suspension; however, the best results are achieved with one- or two-base

see phosphate and sulfate solutions obtained. Is the The anion concentration is too low (for example below about 100 mM), the suspension will not gel.

The intermediate matrices produced in the process of the invention are collected and used for removal washed the excess gelling solution. The matrices are then coated with a coating or crosslinking solution of a polyanionic and preferably polycarboxylated polymer. A preferred crosslinking solution is a 1% poly-L-aspartic acid or poly-L-glutamic acid solution, those in a ratio of 1:15 with 150 mM sodium chloride to a final polymer concentration is diluted from 6.6 χ 10 ~ g / 100 ml. The molecular weight of the poly-L-aspartic or poly-L-glutamic acid is in the range of 3,000 to 100,000 Daltons or greater, with the range of 25,000 to 60,000 Daltons being preferred is. A reaction time of 3 to 6 minutes at room temperature is satisfactory.

The invention is illustrated by the following examples.

example 1

A stock solution of 1.4 % chitosan (CSN-Sigma Chemical Co.) in 14 to 17 mM (mmol / l) isotonic phosphate buffered saline (PBS), prepared as indicated above, was used to determine the optimal CSN concentration for the formation of the intermediate matrix , whereby different chitosan concentrations were investigated. 1 ml samples were taken from the CSN stock solution and mixed with fetal calf serum (FCS-Flow Laboratories)

offset. The droplets were formed and incorporated into the solutions described below using a droplet forming device as described above. Table 1 shows three different chitosan concentrations, namely 0.6, 0.8 and 1 % (weight / volume).

sample

, ml Tabel 1 43 CSN 57 58 O, 42 FCS, ml 28 O, 72 0, O, O,

CSN concentration (w / v)

0.8 I 0.6 % 1.0 i

Two gelling buffers were prepared, namely 110 mM sodium citrate (Sigma) and 125 mM disodium hydrogen phosphate (Sigma). In all samples were the intermediate matrices formed with these buffer solutions acceptable, but with the sodium citrate buffer it was Gelling of sample 1 is poor. While with all samples acceptable intermediate matrices in disodium hydrogen phosphate buffer Sample 3 gave the best gelation.

A 1 liter solution of poly-L-aspartic acid (molecular weight: 40,000 daltons; Sigma) was 1:15 with 150 mM sodium chloride (Sigma) to a concentration diluted from 6.6 χ 10 ~ g / 100 ml of solution. The intermediate matrices formed with Sample 3 were made from removed from the gelling buffer, repeated with isotonic PBS

3432H3

washed and put in the poly-L-aspartic acid solution again suspended. After a reaction time of 6 min at room temperature in the poly-L-aspartic acid solution the cross-linking completely, permanent membranes were created. The capsules were essentially spherical in shape with a diameter of about 380 to 480 μm. Some capsules had small tails. The capsules made in this way were not sticky and had no tendency to clump. The porosity of the capsules has been empirically determined to be about 80,000 Daltons. The example shows that in the method according to the invention acceptable capsules can be formed.

Example 2

It should be shown that encapsulation does not affect cell viability. The cell culture used in this experiment was a Friend erythroleukemia cell line CFEL ₇₄₅ ), ie a mouse erythroleukemia cell line which grows rapidly in a suspension culture.

A culture of FELy.r cells was centrifuged for 5 min; the resulting cake was resuspended in fetal calf serum (Flow Labs) as a slurry. A stock solution of 1.4% chitosan as described above was prepared and mixed with the cell culture solution, the final concentration of chitosan being 1 % (weight / volume) and of cells being about 5 × 10 cells / ml. Intermediate matrices were produced by forcing the chitosan-cell mixture through a droplet-forming device (as described above) and the resulting

liquid microspheres were treated with a 125 mM phosphate solution (as described above). The resulting intermediate matrices were washed repeatedly with isotonic PBS and resuspended in a poly-L-aspartic acid solution (with a molecular weight of 40,000 Dalton) in a concentration of 6.6 10 g / 100 ml in 150 mM sodium chloride. After a reaction time of 6 min at room temperature in the poly-L-aspartic acid solution, the resulting capsules were washed twice with the culture medium RPMI-1640 (Flow Labs), which contains 10 l of fetal calf serum and antibiotics. The microcapsules were introduced into tissue culture flasks with the culture medium and ^{incubated at 37 °} C. and a 5 2 CO ₂ atmosphere.

Samples of the cell culture were taken at various times and examined in a microscope, whereby found was that the cells grow and reproduce, which increases the viability of the cells after Encapsulation proves. It should be noted that the reliquefaction stage has not been applied, since Friend cells can grow in both gel and suspension cultures. Many types of cells however, require a liquid culture medium for reproduction. The viability of the cell should be reduced by the Redissolution of the interior of the capsule not affected will. The capsule provides a micro-environment free of external contamination.

Other modifications or changes will occur to those skilled in the art Embodiments of the described method and the Product within the scope of the invention. To the-

accordingly, other embodiments are also included the requirements.

Claims (30)

Expectations 1. Method for encapsulating a material to be enveloped in a semipermeable membrane, A. the material to be enveloped in an aqueous medium in which a polysaccharide containing cations is dissolved, is suspended, B. the suspension is formed into droplets containing the material to be enveloped, C. the droplets are treated with a solution containing anions, whereby they become discrete, form stable intermediate matrices gel, and D. Treating the intermediate matrices with a polymer containing anions that react with the cations The surface layers of the intermediate matrices are crosslinked to form capsules around the droplets will. 2. The method according to claim 1,
characterized, that the polysaccharide containing cations is an aminated Is polysaccharide. 0192-DBH-419-K1-E 3. The method according to claim 1 or 2, characterized in that the aminated polysaccharide is chitosan. 4. The method according to any one of claims 1 to 3, characterized in that that the anions are selected from phosphate, dibasic phosphate and / or sulfate. 5. The method according to any one of claims 1 to 4, characterized in that that the solution containing anions is prepared by dissolving a salt of the anion in an aqueous solution will. 6. The method according to any one of claims 1 to 5, characterized in that that the anion-containing polymer contains carboxyl groups. 7. The method according to any one of claims 1 to 6, characterized in that that the polymer containing carboxyl groups is selected from polyaspartic acid and / or polyglutamic acid and / or its salts is selected. 8. The method according to any one of claims 1 to 7, characterized in that that it contains an additional step in which the gel is redissolved within the capsule. 343214 9. The method according to any one of claims 1 to 8, characterized in that that the gel was made soluble again by treating the capsule with a solution of a low molecular weight polycation will. 10. The method according to any one of claims 1 to 9, characterized in, that the low molecular weight polycation is selected from spermadine, spermine and / or urea. 11. The method according to any one of claims 1 to 10, characterized in, that the material to be enveloped is selected from enzymes, antibodies, hormones and live cells. 12. The method according to any one of claims 1 to 11, characterized in that that the living cells comprise a tissue culture or their individual cells and the aqueous medium comprises a tissue culture medium is. 13. The method according to any one of claims 1 to 11, characterized in, that the living cells comprise genetically modified cells. 14. The method according to claim 1 for the encapsulation of living cells in a semipermeable membrane, characterized in that - 4 - 3432U3 a. the cells in an aqueous medium that increases viability of the cells is not affected and contains an aminated glucopolysaccharide, suspended will, b. the suspension is formed into droplets containing the cells, c. the droplets with an aqueous ^ anions containing Gelling solution are treated, whereby they form stable, water-insoluble intermediate matrices gel, and d. treating the intermediate matrices with a polymer containing a plurality of carboxyl groups the surface layer of the intermediate matrices permanently becoming a membrane around the droplets is networked. 15. The method according to claim 1 or 14, characterized in that that it contains an additional step in which the interior of the membrane is made soluble again by treatment of the product of stage d. with a solution of a low molecular weight polycation that converts the anions in a practically Precipitation-free reaction removed from the gel. 16. The method according to any one of claims 1, 14 and 15, characterized in that that the gelling solution is an aqueous solution of the salt of an anion selected from phosphate, dibasic Phosphate and / or sulfate. 17. The method according to any one of claims 1, 14 to 16, characterized in that 343214: that the polymer containing a large number of carboxyl groups is selected from polyaspartic acid and / or polyglutamic acid and / or one of their salts is selected. 18. The method according to any one of claims 1, 14 to 17, characterized, that the low molecular weight polycation is selected from spermadine, spermine and / or urea. 19. The method according to any one of claims 1, 14 to 18, characterized in that that the live cells comprise human and animal tissue cells and the aqueous medium comprises a tissue growth medium is. 20. The method according to any one of claims 1, 14 to 18, characterized in that that the living cells comprise genetically modified cells and the aqueous medium is a growth medium. 21. capsule,
marked by a membrane surrounding a closed intracapsular space consisting essentially of an inner layer of a polyaminated polymer and an outer layer made of a polyanionic polymer created by ionic interaction between the aminium groups of the polyaminated polymer and the anions of the polyanionic polymer are crosslinked to form a water-insoluble permeable capsule, produced according to one of claims 1 until 20. - 6 - 3432H3 22. Capsule according to claim 21, characterized in that the polyaminated polymer is an aminated polysaccharide is. 23. Capsule according to claim 21 or 22, characterized in that the aminated polysaccharide is a polyaminated Is glucopolysaccharide. 24. Capsule according to one of claims 21 to 23, characterized in that that the polyanionic polymer includes a polycarboxylated polymer. 25. Capsule according to one of claims 21 to 24, characterized in that that the polyanionic polymer is polyaspartic acid or one of its salts. 26. Capsule according to one of claims 21 to 24, characterized in that that the polyanionic polymer is polyglutamic acid or one of its salts. 27. Capsule according to one of claims 21 to 26, characterized in that that it contains a cell in its culture medium in the intracapsular space. 28. Capsule according to one of claims 21 to 27, characterized in that that the polyaminated polymer and the polyanionic polymer are physiologically compatible with the cell. 29. Capsule according to one of claims 21 to 28, characterized in that that the cells comprise eukaryotic cells. 30. Capsule according to one of claims 21 to 28, characterized in that that the cells comprise genetically modified cells.