CA1144010A

CA1144010A - Process for preparing semi-permeable microcapsules by dilution of a hydrophobic continuous phase with a solvent of different polar character

Info

Publication number: CA1144010A
Application number: CA000333335A
Authority: CA
Inventors: Franklin Lim; Richard D. Moss
Original assignee: Damon Corp
Current assignee: Damon Corp
Priority date: 1978-08-04
Filing date: 1979-08-03
Publication date: 1983-04-05
Also published as: DK151212B; NO148320B; NO792558L; NO148320C; FR2432337A2; FR2432337B2; IT1118818B; DK329179A; SE7906567L; CH648216A5; IT7968610A0; SE434470B; DE2931651A1; DK151212C

Abstract

ABSTRACT OF THE DISCLOSURE

Techniques for producing semipermeable microcapsules by interfacial polymerization are disclosed. The material to be encapsulated and a hydrophilic monomer are emulsified within a hydrophobic continuous phase. Polymerization is initiated by dissolving a second monomer in the continuous phase, and occurs only at the interface of the emulsion to result in the forma-tion 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. This step is accomplished by mixing a second solvent 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.

Description

4~0 1 BACKGROUND OF T~IE-INVENTION
This invention reiates to an encapsulation process and more particularly to a process for producing semipermeable microcapsules.
Canadian Patent ~pplication Serial ~umber 264,735 filed November 2, 1976, discloses a novel technique fGr encap-sulating chemcially active materials in microcapsules whose uniformity of structure and permeability are controlled to an improved degree such that relatively low molecular weight sub-stances with which the encapsulated substance can react candiffuse through the capsule membranes, yet passage of the en-capsulated substance is prevented. The techniques of this application, in addition to providing an improved degree of control over the permeability of the capsule membranes, also enable encapsulation of easily denatured materials such as enzymes and various antibodies such that they remain biochemi-cally operative. This microencapsulation procedure constitutes 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 dis-continuous phase forms a polymeric membrane with a second, complementary monomer dissolved in the continuous phase.
SUMMARY OF THE INVENTION ~
A microencapsulation technique has now been discovered which may be used to encapsulate essentially any core material within membranes having an upper limit of permeability within a selected range, 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 ;' LQl~

1 polycondensation or polyaddition reaction with a second, hydrophobic, complementary monomer is dissolved in water to-gether with the material (if any) to be encapsulated, and the solution is emulsified wi-thin a hydrophobic solvent. When a portion of the complementary monomer is dissolved in the con-tinuous phase of the emulsion, membrane formation begins as interfacial polymerization takes place about the droplets of the discontinuous phase.
In accordance with the invention, the polymerization reaction is allowed to continue only until macroporous, poorly formed capsule membranes are produced, and 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 so that further polymerization occurs preferentially 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 varyin~ the affinity of the continuous phase for the monomers 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.
In one embodiment, the continuous phase at the outset has 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 sta~e of poly~
merization, the continuous phase is altered to have a low 1 affinity for the first monomer, resulting in the precipitation of polymer preferentially within the voids of the raw capsules.
In a preferred embodiment, the continuous phase in the first stage 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, addi-tional quantities of first monomer are drawn through the initially formed membranes and made available for reaction with further quantities of second monomer. In both embodiments, 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., 0-5%, of a multifunctional cross-linking substances 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 hydro-phobic monomer, which terminate polymer chain formation.
~; Two techniques for varying the affinity of the con-tinuous phase for the encapsulated monomers have been employed with success. As disclosed in Canadian Application Serial Number 264,735, the partly formed first stage microcapsules can be separated from the two-phase system and resuspended in a fresh continuous phase of a solvent or solvent system having a -;~ polarity different from the originally employed continuous phase. In accordance with this invention, a continuous phase-miscible solvent is used to dilute the original continuous ,: .
' ' 4~

1 phase to vary its net pola~ity. 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 activ-ity. Thus, a buffered solution having a pH suitable for maintenance of the antibody, etc., often including a stabiliæ-ing carrier such as polyvinyl pyrrolidone, albumin, or dextran, may be used as the discontinuous droplet phase.
In a preferred process, polyamide microcapsules are produced from a hydrophilic monomer comprising a multifunc-tional amine and a hydrophobic monomer comprising a difunction-al acid halide. The amine can comprise a difunctional monomer mixed with from 0-50% 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 amine used in the aqueous discontinuous phase, the lower the permeability limit.
Preferred amines include l,~-hexane diamine, 2,5-dimethyl-piperazine, 1,4-butane diamine, and propylene diamine.
Preferred difunctional acid halides include terephthaloyl 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; dilu-tion with chloroform results in a mixed solvent of increased affinity for amine.
Accordingly, it is an object of the invention to provide a process for producing semipermeable microcapsules useful as a chromotographic separation material. Another object is to encapsulate chemically inactive materials and operable 4!~V

1 biologically and chemically active materials. Still other objects are to provide a method of controlling capsule membrane permeability to an improved degree and a method which may be practiced using a large variety of monomers which react to form polymeric chains by polycondensation or polyaddition.
To this end, in one o its aspects, the invention provides a process for producing microcapsules comprising membranes having an upper limit of permeability within a selected range, said process comprising the steps of:
1~ A. forming a two-phase system comprising ~a hydrophobic continuous phase and a discontinuous phase of discrete aqueous drop-lets containing a first hydrophilic monomer capable of forming a polymer by reaction with a second, complementary hydrophobic monomer;
B. dissolYing 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.
These and other objects and features of the in~ention will be apparent from the following description of some pre-~erred embodiments.

DESCRIPTION OF THE PREFERRED EMBODI~NT
The process of the invention involves a novel 1 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. The size of the drop-lets determines the size of the microcapsules that will be produced. Emulsification can be effected by any of the well-known emulsification techniques such as, for example, using ablender, and is usually done with the aid of an emulsifing agent. Since the size of the discontinuous phase droplets produced in any given technique and thus the size of the result-ing 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 dia-meter. For a detailed disclosure of the method of varying capsule size, reference should be made to Artificial Cells, Thomas M.S. Chang, Chapter. 2.
~ When droplets of a selected size have been produced, a second hydrophobic monomer, soluble in the continuous phase, and capable of forming a polymer by polycondensation or poly-addition with the first monomer is introduced into the suspen-sion. 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 polymeriza-tion is terminated at an :, .~

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1 early stage ~hen the membranes are incompletely forme~, 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 in-capable of confining even high molecular weight materials. On the other hand, if the polymerization is allowed to go to completion, dense, substantially impermeable microcapsules are produced.
In accordance with the invention, the permeability and uniformity of the microcapsule membranes are controlled to an improved degree by varying the affinity of the continuous phase for the discontinuous phase monomer during the course of polymerization. Thus, the thickness of the interface and the amount of first monomer which is available for reaction with the complementary monomer in the continuous phase can be con-trolled to result in membranes having a relatively uniform permeability. Further, within limits, it is possible to tailor the membranes such that they only allow diffusion of molecules below a selected molecular weight, generally within the range of 200 to 30,000 daltons, and are impermeable to higher molecular weight materials.
In one preferred embodiment, the continuous phase in the first polymerization stage is selected to have a low affinity for the 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 for the first monomer is increased so that additional quantities of the monomer permeate the initially formed membrane layer, one or more additional ~4~10 1 layers of polymer are formed about the first, and irnperfections in the first layer are filled in.
; An another embodiment, the affinity of the continuous phase for the first monomer is relatively high at the outset, resulting in the Eormation of a relatively thick, sponge-like polymer framework. In a second stage, the affinity of the continuous phase for the first monomer is decreased so that further polymerization occurs preferentially within the struc-ture of the initially deposited polymer network, filling in the voids and resulting in uniform capsules.

Two methods of varying the affinity of the continuous phase for the first monomer are contemplated. Thus, as dis-; closed in Patent Application 264,735, the raw capsules may be isolated by, for example aspiration of the continuous phase and washing, and then resuspended in a fresh quantity of a solvent of diEferent polarity. Further polymerization is then initiated by dissolving, in some cases incrementally, additional quanti-ties of second monomer in the fresh continuous phase to complete the interfacial polymerization. In the method of -this invention the affinity of the continuous phase for the first monomer is increased or decreased as desired by diluting the continuous ~hase with a solvent, miscible with the originally employed 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 made in accordance with the invention 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. ~nother 1 important feature of the process of the invention is its in-herent ability to overcome the effect of side reactions between the second monomer and water present at the interface. Such reactions form monofunctional monomers which can prematurely terminate polymer chains and disrupt membrane formation. The concentration oE these materials at the interface is limited in the two-stage procedure of the invention.
; In a preferred reaction system, a multifunctional amine and a high molecular weight, hydrophilic filler material such as polyvinyl pyrrolidone, albumin, dextran, or polyethyl-ene glycol is included in the aqueous phase. The filler material serves to prevent collapse of the finally formed ; microcapsules. The continuous phase, at the 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 monomers. 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 soluable monomers. Con~ersely, at the outset the continuous phase can comprise a chloroform-rich 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 _ g_ :'~

1 acid halides may also be used. It is also within the scope o~
the invention to use a polyfunctional first or second monomer together with the difunctional monomers so that a certain amount of cross-linking occurs during formation of the membrane. In general, the inclusion of monomers which result in the forma-tion of cross-links has the effect of lowering membrane per-meability.
The Eoregoing reaction system is disclosed merely by way of example. Thus, various aliphatic, alicyclic, and aromatic hydrocarbons may be used for the nonpolar 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 w~th 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 respecti~e solvents must be of the type which do not interfere with the polymerization reaction between the 29 two or more complementary monomers employed; and

3. there must be available a solvent of a polarity distinctly different from that empIoyed in the continuous phase of the first stage reaction. This solvent must be miscible with the contin~ous phase, so that its polar character can be significantly ~aried.
The criteria for selecting a polymer system for use in the process are as follows:
1. one of the two monomers must be hydrophilic and its complementary monomer must be hydrophobic;
2. the two monomers must spontaneously react on 1 contact to form polymer chains insoluble in both phases; 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 aspect of the inven-tion that some hydrolysis of the hydrophobic monomer can be tolerated without seriously affqcting the quality of -the membrane. The local concentration of hydrolyzed monomer can be minimized by adding monomer to the continuous phase in in-crements.
Polycondensation reactions are well suited in the process of the in~ention, but polyaddition reactions may also be employed. By astute selection of solvents, chosen in accord-ance with the teachings herein to suit particular polymer systems and particular 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 polysulfonamide from a difunctional sulfonyl halide and a diamine. Encapsul-ation pro-cedures using other polyaddition reactions, such as the type disclosed in the Kan et al. U.S. Patent Number 3,864,275 are also within the scope of this invention.
The invention will be further understood from the following nonlimiting examples.
Example 1 One and one-half milliliter of an aqueous carrier solution comprising polyvinyl pyrrolidone, albumin, and 250~uR

., 1 of antisera to thyroxine are mixed with 50~u~ of 0~5M tetra-ethylenepentamine carbonate (pH = 8.2 - 8.6 buffered with CO2).
The aqueous phase is then added to 15 mQ of cyclohexane containing 3~ - 6% ~RLACEL~(sorbitan oleate) as an emulsifier.
The two-phase system is emulsified by means of à magnetic stirring bar, and as stirring continues, one 2 mQ portion of

4:1 (v/v) cyclohexane-chloroform solution containing 0.1 mg/mQ
terephthaloyl chloride is added to initiate polymerization.
Sixty seconds later, another 0.8 mQ of the tereph-thaloyl chloride solution is added. After 60 more seconds,0.5 mQ of pure chloroform are added to increase the affinity of the continuous phase for the pol.yfunctional amines; then, at.30 second intervals, three additional 0.5 mQ increments of pure chloroform are added.
After a total reaction time of four minutes, the emulsion is gently contrifuged and the supernatant liquid dis-carded. The microcapsules are washed with pure cyclohexane and a 50~ aqueous l'WEEN-20 solution (sorbikan monolaurate) buffered to neutral pH with 0.3M Na HCO3.
~0 The foregoing procedure results in capsules having a permeability sufficient to allow passage of thyroxin, (molecular weight 777 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 mQ of an aqueous carrier solution comprising polyvinyl pyrrolidone, albumin, Na2CO3/ NaHCO3 buffer, and 0.3 mQ of glucose oxidase are mixed with 1.2 mQ of hexanediamine carbonate (2.5~; pH 8.4 - 8.6). This aqueous phase is then added to 30 mQ of a mixed organic solvent 1 consisting of 50 parts cyclohexane, 5 parts chloroform, ana 3%-5% sorbitan oleate as an emulsifier. The two-phase system is emulsified by means of an emulsifying stirring probe.
~ ile stirring, 2.6 mQ of the terephthaloyl chloride solution of E~ample 1 is added to initiate polymerization.
Another 0.8 mQ aliquot of the terephthaloyl chloride solution is added 30 seconds later. This is followed by the addition of four 5.0 mQ volumes of cycloheY~ane, spaced at 30 second inter-vals.
io At the end of 3.5 minutes of total polymerization re-action time, the reaction is terminated and the microcapsules harvested as set forth in Example 1. Glucose oxidase is retain-ed within the capsules, yet glucose (MW ~ 180) diffused through the membranes.
Example-3 A 4.0 mQ aqueous phase comprising 1.25 M hexanediamine carbonate and lactate dehydrogenase are emulsified in 20 mQ 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 75juQ
of the diisocyanate is added by means of an infusion pump over a period of S~ minutes as a 5.0 mQ aliquot of solution consist-in~ of 20% cyclohexane ~ lQ% 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 stirrea for an additional 20 minutes. Two minutes before isolating the capsules, the tackiness of the s-urface of ~hé membranes is reduced by adding 0.6 mQ 10%
terephthaloyl chloride. These capsules are permeable to sub-stances in the-I~olecular weight range below-about 1000 daltons.

4~
:;~
1 Example 4 .
~ Iexanediamine carbonate (p~ = 8.5 ~ 0.1) solution is prepared by mixing 17.7 mQ 1,6-hexanediamine with 32 mQ of water, and bubbling CO2 through the solution for about 1 hour or until the pH level is reached. Terephthaloyl chloride (TCl) solution is prepared by adding 20 g TCl in 200 mQ of organic solvent consisting of ~ parts cyclohexane and 1 part chloroform.
TCl is dissolved by stirring vigorously, and the solution is then centrifuged for 10 minutes at 2600 rpm. Any precipitate 1 n is discarded.
750 mQ cyclohexane are mixed with 125 m~ SP~N~85 in a 2-liter mixer equipped with a magnetic stirring bar. While stirring, a mixed solution made from 25 mQ of 15% polyvinyl-pyrrolidone - 4% bovine serum albumin, 40 mQ of phosphate buffered saline premixed with 5 mQ of antiserum, and 30 mQ of hexanediamine carbonate solution is added to the cyclohexane.
When droplets of the desired size have been produced, 70 mQ TCl solution are added. Thirty seconds later, 37.5 mQ of TCl are added. Sixty seconds later, 25 mQ of chloroform are added, and three additional 25 mQ ali~uots 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 - 30 membranes. Although the disclosure describes a preferred embodiment of the invention, it is to be understood the inven-tion is not restricted to this particular embodiment.

- ~ t~d~ 14-. . .

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for producing microcapsules comprising membranes having an upper limit of permeability within a select-ed 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 drop-lets 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 continu-ous 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 of claim 1 wherein, in the two-phase system of step A, the continuous phase 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 of 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 of claim 3 wherein the polar solvent is added in increments over the course of the polymerization reaction.

5. The process of claim 1 wherein, in the two-phase system of step A, the continuous phase 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 of claim 1 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 of claim 6 wherein the filler material is selected from the group consisting of polyvinylpyrrolidone, polyethylene glycol, polysaccharides, and albumin.

8. The process of claim 1 wherein the first monomer is selected from the group consisting of multifunctional alcohols and amines and the second monomer is selected from the group consisting of diacid halides, diisocyanates, and difunctional sulfonyl halides.

9. The process of claim 1 wherein the first monomer is selected from the group consisting of 1,6-hexanediamine, tetraethylenepentamine, and mixtures thereof.

10. The process of claim 9 wherein said second monomer is selected from the group consisting of terephthaloyl chloride, sebacyl chloride, and mixtures thereof.

11. The process of claim 1 wherein the first monomer is a multifunctional amine and the second monomer is a diacid halide.

12. The process of claim 1 wherein second monomer is added in increments during the course of the polymerization reaction.