GB2026976A - Process for preparing semipermeable microcapsules - Google Patents

Abstract

Semipermeable microcapsules are made by an interfacial polymerization process in which the material to be encapsulated and a hydrophilic monomer are emulsified within a hydrophobic continuous phase and then polymerization is initiated by dissolving a second monomer in the continuous phase. Polymerization occurs only at the interface of the emulsion droplets and results in the formation of macroporous, poorly defined capsule membranes. Next, the affinity of the continuous phase for the hydrophilic monomer is varied by altering the polarity of the continuous phase using a second solvent which is mixed with the continuous phase. By controlling the affinity and the concentration of the second monomer, it is possible to produce microcapsules having uniform capsule membranes and a selected upper limit of permeability. Microcapsules prepared by the process are useful for separating materials of differing molecular weight and for chemical and biological assays.

Description

SPECIFICATION Process for repairing semipermeable microcapsules This invention relates to an encapsulation process and more particularly to a process for producing semi permeable microcapsules.

Our British Patent No. 1,540,461 discloses a technique for encapsulating chemically active materials in microcapsules whose uniformity of structure and permeability are controlled to an improved degree such that relatively low molecular weight substances with which the encapsulated substance can react can diffuse through the capsule membranes, yet passage of the encapsulated substance is prevented.

The technique in addition to providing an improved degree of control over the permeability of the capsule membranes, also enables easily denatured materials such as enzymes and various antibodies to be encapsulated such that they remain biochemi cal ly operative. The microencapsulation procedure embodies an improvement over the well-known interfacial polymerization technique which utilizes the interface of an emulsion as a reaction zone wherein a first monomer solubilized in the discontinuous phase forms a polymeric membrane with a second, complementary monomer dissolved in the continuous phase.

A microencapsulation technique has now been developed which may be used to encapsulate essentially any core material within membranes having an upper limit of permeability within a selected range.

According to the present invention, there is provided a process for producing microcapsules comprising membranes having an upper limit of permeability within a selected range, said process comprising the steps of: A. forming a two-phase system comprising a hydrophobic continuous phase and a discontinuous phase of discrete aqueous droplets containing a first hydrophilic monomer capable of forming a polymer by reaction with a second, complementary hydrophobic monomer; B. dissolving a portion of said second monomer in the continuous phase to effect interfacial polymerization about the droplets of the discontinuous phase; C. altering the affinity of the continuous phase for said first monomer by changing the polarity of the continuous phase by dilution with a solvent of different polar character;; D. allowing further polymerization to occur at the interface of the altered continuous phase; and E. terminating the interfacial polymerization when microcapsules of the selected permeability have been produced.

The invention is hereinafter described in more detail by way of example.

The permeability of the microcapsules is determined during membrane formation by controlling certain parameters of the interfacial polymerization reaction. Briefly, a first, hydrophilic monomer capable of forming a copolymer by polycondensation or polyaddition reaction with a second, hydrophobic, complementary monomer is dissolved in water together with the material (if any) to be encapsulated, and the solution is emulsified within a hydrophobic solvent. When a complementary monomer is dissolved in the continuous phase of the emulsion, membrane formation begins as interfacial polymerization takes place about the droplets of the discontinuous phase.

In practising the invention, the polymerization reaction is allowed to continue only until macroporous, poorly formed capsule membranes are produced. Then, in a second stage, the affinity of the continuous phase for the first monomer contained in the discontinuous phase droplets is varied by altering the polarity of the continuous phase. Further polymerization occurs preferentially either within the macroporous capsule membranes, or in a second, outer layer. Finally, the polymerization is terminated when microcapsules of the selected upper limit of permeability have been produced. The technique of varying the affinity of the continuous phase for the monomer dissolved in the discontinuous phase droplets enables one to exercise a degree of control over the thickness of the interface and thus over the site of polymer formation.Further, it allows one to minimize side reactions between continuous phase-solubilized monomers, e.g., diacid chlorides, and water in the discontinuous phase.

The continuous phase at the outset could have a relatively high affinity for the encapsulated monomer so that a relatively thick polymer network is produced about the droplets where the monomers meet. In a second stage of polymerization, the affinity of the continuous phase for the first monomer is reduced, resulting in the precipitation of polymer preferentially within the voids of the raw capsules. In a preferred way of practising the invention, however, the continuous phase initially has a low affinity for the encapsulated monomer so that a thin polymer membrane is produced at the interface and in the second stage, the continuous phase is altered to have a relatively high affinity for the first monomer.

Thus, additional quantities of first monomer are drawn through the initially formed membranes and made available for reaction with further quantities of the second monomer. In either mode of practising the invention, the upper limit of permeability can be varied with improved precision by controlling the duration of the first and second stage reactions, the polarity of the continuous phase, the concentrations of the monomers, and by including small amounts, e.g., 0 to 5% of a multifunctional cross-linking substance with one of the monomers.

The complementary monomer which is soluble in the continuous phase is preferably added in increments over the course of the reaction. This results in a lessening of side reactions, between water from the droplet phase and the hydrophobic monomer, which terminate polymer chain formation.

According to this invention a continuous phasemiscible solvent is used to dilute the original continuous phase to vary its net polarity. If the material sought to be encapsulated is easily denatured, e.g., an antibody or an enzyme, the pH of the discontinuous phase is controlled so that the labile material retains much of its biological activity. Thus, a buf fered solution having a pH suitable for maintenance of the antibody, etc., often including a stabilizing car rier such as polyvinyl, pyrrolidone, albumin, or dex tran, may be used as the discontinuous droplet phase.

In a preferred process embodying the invention, polyamide microcapsules are produced from a hyd rophilic monomer comprising a multifunctional amine and a hydrophobic monomer comprising a difunctional acid halide. The amine can comprise a difunctional monomer mixed with from 0 to 50 /O of a polyfunctional cross-linker, e.g., tetraethylenepentamine, although successful microencapsulations have been done using only pentamines. In general, the higher the concentration of polyfunctional amime used in the aqueous discontinuous phase, the lower the permeability limit. Preferred amines include 1,6 hexane diamine, 2, 5-dimethylpiperazine, 1,4 butane diamine, and propylene diamine. Preferred difunctional acid halides includeterephthaloyl chloride and sebacyl chloride.For the foregoing polymer systems, the preferred continuous phase solvents comprise cyclohexane, diluted or mixed with chloroform as appropriate. The affinity of pure cyclohexane for the amines is low; dilution with chloroform results in a mixed solvent of increased affinity for amine.

Preferred embodiments of the invention will now be described by way of example.

The process according to the invention involves a variation in the well known process for microencapsulation known generally as interfacial polymerization. This technique utilizes a pair of mutually immiscible solvents or solvent systems, one being hydrophobic, and the other being water. The material to be encapsulated and a first, hydrophilic; monomer are dissolved in water, and the solution is emulsified to form an aqueous, discontinuous or droplet phase dispersed in the hydrophobic, continuous phase. The size of the droplets determines the size of the microcapsules that will be produced.

Emulsification can be effected by any of the wellknown emulsification techniques, for example by using a blender, and usually with the aid of an emulsifying agent. Since the size of the discontinuous phase droplets produced in any given technique and thus the size of the resulting capsules will vary within a specific range, one or more filters may be used to separate oversized or undersized capsules made in any given run to minimize differences in capsule diameter. For a detailed disclosure of the method of varying capsule size, reference should be made to Artificial Cells, Thomas M. S. Chang, Chapt.

2.

A second, hydrophobic, monomer is introduced into the suspension when droplets of a selected size have been produced. The second monomer is solu ble in the continuous phase and is capable of form ing a polymer by polycondensation or polyaddition with the first monomer. Polymerization occurs only at the interface of the two-phase system where the complementary monomers meet. The monomers must be chosen from among those which exhibit suitable solubility properties in the solvents selected.

Utilizing this prior art technique, one can exert only crude control on capsule membrane quality, uniformity, and permeability. Thus, if polymerization is terminated at an early stage when the membranes are incompletely formed, the resulting microcapsules have widely varying permeability and are typically characterized by a high frequency of macroporous defects where little or no polymerization has occurred. The result is a quantity of microcapsules, many of which are incapable of confining even high molecular weight materials. On the other hand, if the polymerization is allowed to goto completion, dense, substantially impermeable microcapsules are produced.

We now control the permeability and uniformity of the microcapsule membranes to an improved degree by varying the affinity of the continuous phase for the discontinuous phase monomer in the course of polymerization. Varying the affinity enables the thickness of the interface and the amount of the first monomer which is available for reaction with the complementary monomer in the continuous phase to be controlled; membranes having a relatively uniform permeability can be achieved. Further, within limits, it is possible to tailor the membranes such that they only allow diffusion therethrough of molecules below a selected molecu larweight, generally within the range of 200 to 30,000 daltons, and are impermeable to higher molecular weight materials.

In one embodiment, the continuous phase in the first polymerization stage is selected to have a low affinityforthe first monomer. This results in the formation of a thin membrane in a narrow interface zone where the complementary monomers come into contact. In a second stage, the affinity of the continuous phase forthe first monomer is increased so that additional quantities of the monomer permeate the initially formed membrane layer, one or more additional layers of polymer are formed aboutthe first, and imperfections in the first layer are filled in.

In a second embodiment, the affinity of the continuous phaseforthefirst monomer is relatively high at the outset, resulting in the formation of a relatively thick, sponge-like polymer framework. In the second stage, the affinity of the continuous phase for the first monomer is decreased so that further polymerization occurs preferentially within the structure ofthe initially deposited polymer network, filling in the voids and resulting in uniform capsules.

In accordance with this invention, the affinity of the continuous phase for the first monomer is increased or decreased as desired by diluting the continuous phase with a solvent, miscible with the originally employed continuous phase solvent, which progressively varies the net polarity of the continuous phase.

From the foregoing it will be appreciated that the improved degree of control over the permeability and quality of microcapsules is achieved by varying the nature of the interface during the course of the interfacial polymerization, and that this is made possible by controlling the polarity of the continuous phase. Another important feature of the process according to the invention is its inherent ability to overcome the effect of side reactions between the second monomer and water present at the interface.

Such reactions may form monofunctional monomeres which can prematurely terminate polymer chains and disrupt membrane formation. The concentration of these materials at the interface is limited in the present two-stage procedure.

In a preferred reaction system, a multifunctional amine and a high molecular weight, hydrophilic filler material such as polyvinyl pyrrolidone, albumin, dextran, or polyethylene glycol is included in the aqueous phase. The filler material serves to prevent collapse of the finally formed microcapsules. The continuous phase, atthe outset, consists of a diacid halide dissolved in pure cyclohexane or a solvent system comprising cyclohexane mixed with a small amount of chloroform, both of which have a low affinity for water soluble momomers. The second stage of polymerization is then effected in a continuous phase comprising a cyclohexane based solvent richer in chloroform, which has increased affinity for water soluble monomers.Conversely, at the outset the continuous phase can comprise a chloroformrich cyclohexane solvent system and further polymerization can be conducted in a mixed solvent of decreased chloroform content. This process results in the formation of polyamide microcapsules.

A preferred first monomer is 1, 6 hexane diamine, but many other multifunctional, water soluble amines may be used. Microcapsules having a permeability limit below about 1000 daltons have been made using tetraethylene pentamine as the hydrophilic monomer. Terephthaloyl chloride is a preferred complementary monomer, but others, e.g., sebacyl and azelaic acid halides may also be used. It is also within the scope of the invention to use a polyfunctional first or second monomer together with the difunctional monomers so that a certain amount of crosslinking occurs during formation of the membrane. In general, the inclusion of monomers which result in the formation of cross-links has the effect of lowering membrane permeability.

The foregoing reaction system is merely exemplary. Thus, various aliphatic, alicyclic, and aromatic hydrocarbons may be used for the non-polar component of the continuous phase, and these may be modified as desired with miscible organic solvents containing various polarity imparting moieties. Petroleum ether fractions, mixed as appropriate with halogenated organic solvents may be used. In general, the only requirements for the solvent system are that: 1. mutually immiscible solvents or solvent systems must be used for the continuous and discontinuous phases; 2. The respective solvents must be of the type which do not interfere with the polymerization reaction between the two or more complementary monomers employed; and 3. there must be available a solvent of a polarity distinctly different from that employed in the continuous phase of the first stage reaction.This solvent must be miscible with the continuous phase, so that its polar character can be significantly varied.

The criteria for selecting a polymer system for use in the process are as follows: 1. one of the monomers must be hydrophilic and its complementary monomer must be hydrophobic; 2. the monomers must spontaneously react on contact two form polymer chains insoluble in both phase; and 3. reaction of the selected monomers should be inhibited as little as possible by the presence of the solvents used in the respective phases of the reaction system.

Regarding point 3, it should be noted that some degree of solvent interference, i.e., hydrolysis side reactions, is unavoidable. However, it is an important advantage of the invention that some hydrolysis of the hydrophobic monomer can be tolerated without seriously affecting the quality of the membrane.

The local concentration of hydrolyzed monomer can be minimized by adding monomer to the continuous phase in increments.

Polycondensation reactions are well suited for use in practising the invention, but polyaddition reactions may also be employed. By astute selection of solvents, chosen in accordance with the teachings herein to suit particular polymer systems and materials to be encapsulated, those skilled in the art will be able to produce capsule membranes of, for example, polyester, from a polyol and a diacid halide, other polyamides from diamines and diacid halides, polyurea from diamines and diisocyanates, and polysulfonamidefrom a difunctional sulfonyl halide and a diamine. Encapsulation procedures using other polyaddition reactions, such as the type disclosed in the Kan et al. U.S. Patent No. 3,864,275 are also within the scope of this invention.

The invention will be further understood from the following non-limiting examples.

Example 1 One and one-half milliliter of an aqueous carrier solution comprising polyvinyl pyrrolidone, albumin, and 250 ,al of antisera to thyroxine are mixed with 50 'LI of 0.5M tetraethylene-pentamine carbonate buffered to pH = 8.2 - 8.6 with CO2. The aqueous phase is then added to 15 ml of cyclohexane containing 3% - 6% ARLACEL (sorbitan oleate) as an emulsifier. The two-phase system is emulsified by means of magnetic stirring bar, and as stirring continues, one 2 ml portion of 4:1 (v/v) cyclohexane-chloroform solution containing 0.1 mg/ml terephthaloyl chloride is added to initiate polymerization.

Sixty seconds later, another 0.8 ml of the terephthaloyl chloride solution is added. After 60 more seconds, 0.5 ml of pure chloroform are added to increase the affinity of the continuous (organic solvent) phase for the polyfunctional amines; then, at 30 second intervals, three additional 0.5 ml increments of pure chloroform are added.

After a total reaction time of four minutes, the emulsion is gently centrifuged and the supernatant liquid discarded. The microcapsules are washed with pure cyclohexane and a 50% aqueous TWEEN-20 solution (sorbitan monolaurate) buffered to neutral pH with 0.3M NaHCO3.

The foregoing procedure results in capsules having a permeability sufficient to allow passage of thyroxin, (molecularweight777 daltons) and lower molecular weight materials, yet insufficient to allow leakage of antibody from the interior of the capsules.

Example 2 Two and one-half ml of an aqueous carrier solution comprising polyvinyl pyrrolidone, albumin, Na2CO3/NaHCO3 buffer, and 0.3 ml of glucose oxidase are mixed with 1.2 ml of hexanidiamine carbonate (2.5M; pH 8.4-8.6). This aqueous phase is then added to 30 ml of a mixed organic solvent consisting of 50 parts cyclohexane, 5 parts chloroform, and 3% to 5% sorbitan oleate as an emulsifier. The twophase system is emulsified by means of an emulsifying stirring probe.

While stirring 2.6 ml of the terephthaloyl chloride solution of Example 1 is added to initiate polymerization. Another 0.8 ml aliquot of the terephthaloyl chloride solution is added 30 seconds later. This is followed by the addition of four 5.0 ml volumes of cyclohexane, spaced at 30 second intervals.

At the end of 3.5 minutes of total polymerization reaction time, the reaction is terminated and the microcapsules harvested as set forth in Example 1. Glucose oxidase is retained within the capsules, yet glucose (MW-=180) diffused through the membranes.

Example 3 A4.0 ml aqueous phase comprising 1.25 M hexanediamine carbonate and lactate dehydrogenase are emulsified in 20 ml of pure cyclohexane containing 2% non-ionic surfactant (Arlecel). While stirring vigorously, membrane formation is initiated as toluene diisocyanate is added to the emulsion. A total of 75 'LI of the diisocyanate is added by means of an infusion pump over a period of 8 minutes as a 5.0 ml aliquot of solution consisting of 90% cyclohexane 10% chloroform. The affinity of the continuous phase for the diamine is thus continually increased until all of the cyclohexane-soluble diisocyanate has been added. The system is then stirred for an additional 20 minutes.Two minutes before isolating the capsules, the tackiness of the surface of the membranes is reduced by adding 0.6 ml 10% terephthaloyl chloride. These capsules are permeable to substances in the molecular weight range below about 1000 daltons.

Example 4 Hexanediamine carbonate (pH = 8.5 5 0.1) solu- tion is prepared by mixing 17.7 ml 1,6 hexanediamine with 32 ml of water, and bubbling CO2 through the solution for about 1 hour or until the indicated pH level is reached. Terephthaloyl chloride (TCI) solution is prepared by adding 20 g TCI in 200 ml of organic solvent consisting of 4 parts cyclohex ane and 1 part chloroform. TCI is dissolved by stir ring vigorously, and the solution is then centrifuged for 10 minutes at 2600 rpm. Any precipitate is dis carded.

750 ml cyclohexane are mixed with 125 ml SPAN-85 in a 2-liter mixer equipped with a magnetic stirring bar. While stirring, a mixed solution made from 25 ml of 15% polyvinyl-pyrrolidone-4% bovine serum albumin,40 ml of phosphate buffered saline premixed with 5 ml of antiserum, and 30 ml of hexane-diamine carbonate solution is added to the cyclohexane. When droplets of the desired size have been produced, 70 ml TCI solution are added. Thirty seconds later, 37.5 ml of TCI are added. Sixty seconds later, 25 ml of chloroform are added, and three additional 25 ml aliquots of chloroform are added at 30 second intervals.

The microcapsules are recovered by centrifuging the two-phase reaction system, decanting the supernatant, and mixing the capsules with TWEEN-20 (buffered with NaHCO3) and phosphate buffered saline. The capsules contain polyvinylpyrrolidone and bovine serum albumin as filler materials. Substances having a molecular weight in excess of about 20,000 daltons (such as most antibodies) cannot penetrate the membranes. Substances having a molecular weight below about 5000 daltons penetrate the membranes.

We have disclosed in the foregoing, a process for producing semi-permeable microcapsules useful as a chromotgraphic separation material. The microcapsules can encapsulate chemically inactive materials and operable biologically and chemically active materials. The process promotes control of capsule membrane permeability to an improved degree, and may be practiced using a large variety of monomers which react to form polymeric chains by polycondensation or polyaddition.

Claims (15)

1. A process for producing microcapsules comprising membranes having an upper limit of permeability within a selected range, said process comprising the steps of: A. forming a two-phase system comprising a hydrophobic continuous phase and a discontinuous phase of discrete aqueous droplets containing a first hydrophilic monomer capable of forming a polymer by reaction with a second, complementary hydrophobic monomer; B. dissolving a portion of said second monomer in the continuous phase to effect interfacial polymerization about the droplets of the discontinuous phase; C. altering the affinity of the continuous phase for said first monomer by changing the polarity of the continuous phase by dilution with a solvent of different polar character; D. allowing further polymerization to occur at the interface of the altered continuous phase; and E. terminating the interfacial polymerization when microcapsules of the selected permeability have been produced.

2. The process according to claim 1, wherein the continuous phase of step A has a low affinity for said first monomer so that a thin membrane is produced in step B, and in step C, the affinity of the continuous phase for the first monomer is increased and an additional layer of polymer is produced about the droplets of the discontinuous phase.

3. The process according to claim 2, wherein the affinity of the continuous phase for the first monomer is increased by diluting the continuous phase with a polar solvent.

4. The process according to claim 1,2 or 3, wherein the diluent solvent is added in increments in step C over the course of the polymerization reaction.

5. The process according to claim 1, wherein the continuous phase of step A is selected to have a relatively high affinity of the first monomer so that membranes comprising a thick polymer network are produced in step B, and in step C, the affinity of the continuous phase for the first monomer is decreased so that further polymerization occurs preferentially within the polymer network.

6. The process according to any preceding claim, wherein a substance incapable of traversing the membranes produced in step D is included in the aqueous droplets of step A as a filler material.

7. The process according to claim 6, wherein the filler material is selected from polyvinyl pyrrolidone, polyethylene glycol, polysaccharides, and albumin.

8. The process according to any of the preceding claims, wherein the first monomer is selected from multifunctional alcohols and amines and the second monomer is selected from diacid halides, diisocyanates, difunctional sulfonyl halides and mixtures thereof.

9. The process according to any of claims 1 to 7, wherein the first monomer is a multifunctional amine and the second monomer is a diacid halide.

10. The process according to any of claims 1 to 7, wherein the first monomer is selected from 1,6 hexanediamine, tetraethylenepentamine, and mixtures thereof.

11. The process according to claim 10, wherein the second monomer is selected from terephthaloyl chloride, sebacyl chloride, and mixtures thereof.

12. The process according to any of the preceding claims, wherein the second monomer is added in increments during the course of the polymerization reaction.

13. A process according to claim 1 for producing microcapsules, substantially as herein described.

14. A process for producing microcapsules in accordance with Example 1 or Example 2 or Example 3 or Example 4 herein.

15. Semipermeable microcapsules when produced by the process claimed in any of the preceding claims.