DK151212B - PROCEDURE FOR MANUFACTURING SEMIPERMEABLE MICROCAPLES - Google Patents

Description

in 151212

The present invention relates to an encapsulation method, in particular to a method of producing semipermeable microcapsules.

Norwegian patent no. 147,883 (parallel to Danish patent application 5 no. 4455/76) discloses a new technique for encapsulating chemically active materials in microcapsules, whose uniformity in structure and permeability is regulated in an improved manner so that relatively low molecular weight substances with which the encapsulated substance can be reacted can diffuse through the capsule membranes and yet prevent passage of the encapsulated substance. The technique described there enables, in addition to providing an improved degree of control over the permeability of the capsule membranes, also encapsulation of slightly denatured substances, e.g. enzymes and various antibodies so that they become biochemically operative. This microencapsulation method represents an improvement over the well-known interface polymerization technique utilizing an emulsion interface as a reaction zone in which the first monomer solubilized in the discontinuous phase forms a polymeric membrane with a second, complementary monomer dissolved in it. continuous phase. However, in the method described in the said Norwegian patent it is necessary to isolate the raw capsules obtained in a first step and then resuspend them in a second hydrophobic solvent, in which the first monomer is very little soluble. As more of the second monomer is added to the suspension, further polymerization occurs, preferably within the network formed by the first polymerization. Although this technique led to improved capsule membranes, it has a labor-intensive, two-stage process which was not well-suited for industrial scale use.

A much better microencapsulation technique has now been found which can be used to encapsulate virtually any core material in membranes having an upper limit of permeability in a selected range, by which technique it is possible to change the polarity of the continuous phase in situ , ie the two-step process becomes redundant. The permeability of the microcapsules is determined during the formation of the 35 membranes by regulating certain parameters of interface polymerization. In short, 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 are encapsulated, and the solution is emulsified in a hydrophobic solvent. As a portion of the complementary monomer dissolves in the continuous phase of the emulsion, membrane formation begins as the interface polymerization takes place around the drops in the discontinuous phase.

According to the invention, the polymerization reaction is only allowed to proceed until macroporous, poorly formed capsule membranes are formed, and in a second step the affinity of the continuous phase is varied to the first monomer contained in the drops of the discontinuous phase by changing the polarity of the continuous phase in such a way. further polymerization occurs, preferably within the macroporous capsule membranes, or in another outer layer. Finally, the polymerization is completed when microcapsules with the desired upper limit of permeability have been prepared. The technique of varying the affinity for the continuous phase for the monomers dissolved in the droplets of the discontinuous phase makes it possible to effect some kind of control of the thickness of the interface and thus of the position of the polymer formation. Furthermore, it becomes possible to reduce side reactions between solubilized monomers in the continuous phase, e.g. disyrechlorides, and water in the discontinuous phase. Since the previously required two-stage process is no longer necessary, the loss of solvents at the intermediate isolation step is also avoided.

In one embodiment of the process of the invention, the continuous phase initially has relatively high affinity for the encapsulated monomer, so that a relatively thick polymer network 30 is produced around the droplets at the sites where the monomers meet. In a second step of the polymerization, the continuous phase is changed in such a way that it gets low affinity to the first monomer, resulting in the precipitation of polymer, preferably within the voids of the raw capsules. In a preferred embodiment, the continuous phase of the first stage has low affinity to the encapsulated monomer, so that a thin polymer membrane is produced at the interface, and in the second stage the continuous phase is changed to have a relatively high affinity. opposite the first monomer. Thus, additional amounts of the first monomer are drawn through the membranes initially formed 5 and made available for reaction with additional amounts of the second monomer. In both embodiments, the upper permeability limit can be varied with improved precision by controlling the duration of first and second stage reactions, the polarity of the continuous phase, the concentration of the monomers and by including small amounts, e.g. 0-5%, of a polyfunctional cross-linking substance with one of the monomers.

The complementary monomer that is soluble in the continuous phase is preferably added in portions during the reaction. This results in fewer side reactions between water from the droplet phase and the hydrophobic monomer which terminates the polymer chain formation.

Two techniques for varying the continuous phase affinity for the encapsulated monomers have been successfully used. As disclosed in U.S. Patent No. 4,324,683, the partially 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 initial continuous phase used.

According to the invention, a continuous phase miscible solvent is used to dilute the initial continuous phase to vary its net polarity. If the material sought to be encapsulated is easily denatured, for example. is an antibody or enzyme, the pH value of the discontinuous phase is regulated in such a way that the labile material retains much of its biological activity. Thus, a buffered solution having a pH suitable for maintaining the antibody can often include a stabilizing carrier, e.g. polyvinylpyrrolidone, albumin or dextran, is used as the discontinuous drop phase.

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In a preferred method of the invention, polyamide microcapsules are prepared from a hydrophilic monomer comprising a poly-functional amine and a hydrophobic polymer comprising a difunctional acid halide. The amine may comprise a difunctional monomer mixed with 0 - 50% of a polyfunctional crosslinker, e.g. tetraethylene-pentamine, although microcapsules containing only pentamines have been successfully prepared. It can generally be stated that the higher the concentration of polyfunctional amine used in the aqueous discontinuous phase, the lower the permeability limit. Preferred amines include 1,6-hexanediamine, 2,5-dimethylpiperazine, 1,4-butanediamine and propylenediamine. Preferred difunctional acid halogenides include terephthaloyl chloride and sebacyl chloride. For the above polymer systems, the preferred continuous phase solvents include cyclohexane, diluted or mixed with chloroform. The affinity of pure cyclohexane to the amines is low; Dilution with chloroform results in a mixed solvent with increased affinity for amine.

Thus, it is an object of the process of the invention to provide a process for producing semipermeable microcapsules which can be used as a chromatographic separation material. Another purpose is to encapsulate chemically inactive materials and operable biologically and chemically active materials. Further objects are to provide a process for regulating the capsule membrane permeability to an improved degree and a process that can be practiced using a wide variety of monomers which react to form polymer chains by polycondensation or polyaddition.

These and other objects and features of the invention will become apparent from the description below of some preferred embodiments.

The process of the invention involves a new variation of the well-known method of microencapsulation, which is generally known as interface polymerization. This technique utilizes a pair of mutually immiscible solvents or solvent systems, one of which is hydrophobic and the other of which is water. The material to be encapsulated and a first hydrophilic monomer are dissolved in water and the solution emulsified to form the aqueous, discontinuous or droplet phase. The size of the droplets determines the size of the microcapsules produced. The emulsification may be carried out by any well-known emulsifying technique, e.g. using a mixing apparatus, and is usually carried out by means of an emulsifier.

Since the size of the discontinuous phase droplets produced by any method, and thus the size of the resulting capsules, varies within a specific range, one or more filters may be used for separating too large or too small capsules which are manufactured at any time so that the differences in the capsule diameters are minimized. For detailed description of the method for varying capsule size, see Artificial Cells, Thomas M.S. Chang, Chapter 2.

When droplets of the selected size are prepared, a second hydrophobic monomer which is soluble in the continuous phase and capable of forming a polymer by polycondensation or polyaddition with the first monomer is introduced into the suspension. The polymerization occurs only at the interface of the two-phase system where the complementary monomers meet. Monomers must be selected from those exhibiting suitable solubility properties in the selected solvents.

Using this prior art, only a rough control of the capsule membrane can be exerted on the quality, uniformity and permeability. Therefore, if the 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 having high frequency of macroporous defects at the sites where polymerization or polymerization occurred in only a small amount. degree. The result is a plethora of microcapsules, many of which are unable to hold even high molecular materials. On the other hand, if the polymerization is allowed to proceed until it is complete, dense, virtually impermeable microcapsules are obtained.

According to the invention, the permeability and uniformity of the microcapsule membranes is improved to a greater extent by varying the affinity of the continuous phase with the discontinuous phase monomer during the course of polymerization. Thus, the thickness of the interface and the amount of the first monomer available for reaction with the complementary monomer in the continuous phase can be controlled in such a way as to obtain membranes having relatively uniform permeability. Furthermore, it is possible within certain limits to "tailor" the membranes to allow only diffusion of molecules below a certain molecular weight, generally in the range of 200-30,000 Daltons, and are impervious to higher molecular weight materials.

In a preferred embodiment of the process of the invention, the continuous phase of the first low affinity polymerization step is selected against the first monomer. This leads to the formation of a thin membrane in the narrow interface zone where the complementary monomers contact each other. In a second step, the affinity of the continuous phase towards the first monomer is increased such that additional amounts of monomer penetrate the initially formed membrane layer, one or more additional layers of polymer are formed around the first, and imperfections in the first layer 20 are filled.

In another embodiment, the affinity of the continuous phase against the first monomer is relatively high initially, leading to the formation of a relatively thick, sponge-like polymer network. In a second step, the affinity of the continuous phase against the first monomer is reduced so that further polymerization occurs, preferably within the structure of the initially deposited polymer network, thereby filling the voids, leading to uniform capsules.

Two methods for varying the continuous phase affinity to the first monomer are envisaged. As stated in Norwegian patent no.

147,883, the raw capsules can be isolated by e.g. evaporation of the continuous phase and washing and subsequent resuspending in a fresh amount of solvent of a different polarity. Further polymerization is then initiated by dissolving, in some cases portionwise, additional amounts of the second monomer in the fresh continuous phase to complete the interface polymerization. In the process of the present invention, the affinity of the continuous phase against the first monomer is increased or reduced, as desired, by diluting the continuous phase with a solvent miscible with the initially used solvent, which progressively varies the net polarity. in the continuous phase.

It can be seen from the above that the improved degree of control of the permeability and quality of the microcapsules produced by the method of the invention is achieved by varying the nature of the interface during the interface polymerization, and this is made possible by controlling the polarity of the continuous phase. Another important feature of the process of the invention is its inherent ability to overcome the effect of side reactions between the second monomer and the water present on the interface.

Such reactions form monofunctional monomers that can prematurely terminate polymer chains and interrupt membrane formation. The concentration of these materials at the interface is limited in the two-step process of the invention.

In a preferred reaction system, a polyfunctional amine and a high molecular weight hydrophilic filler, e.g. polyvinylpyrrolidone, albumin, dextran or polyethylene glycol, in the aqueous phase. The filler serves to prevent the finally formed microcapsules from collapsing. The continuous phase initially 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 low affinity for water-soluble monomers.

The second polymerization step is then carried out in a continuous phase comprising a cyclohexane based solvent which is richer in chloroform which has increased affinity for water soluble monomers. Alternatively, the continuous phase may initially comprise a chloroform-rich cyclohexane solvent system, and further polymerization may be carried out in a mixed solvent of reduced chloroform content. This process results in the formation of polyamide microcapsules.

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A preferred first monomer is 1,6-hexanediamine, but other polyfunctional, water-soluble amines can also be used. Microcapsules with a permeability limit of less than approx. 1000 Daltons are prepared using tetraethylene pentamine as the hydrophilic monomer.

Terephthaloyl chloride is a preferred complementary monomer, but others may also be used, e.g. sebacyl and azelaic acid halides. It is within the scope of the present invention to use a polyfunctional first or second monomer with the difunctional monomers so that some degree of cross-linking occurs during the formation of the membrane. In general, incorporation of monomers resulting in the formation of cross-links results in decreased membrane permeability.

The above reaction system is given by way of example only.

Thus, various aliphatic, alicyclic and aromatic hydrocarbons can be used as the non-polar component of the continuous phase, and these can be modified as desired with miscible organic solvents containing various polarity-giving moieties. Petroleum ether fractions may be used suitably mixed with halogenated organic solvents. In general, the only requirements for the solvent system are the following: 1) Mutually immiscible solvents or solvent systems must be used for the continuous and discontinuous phases; 2) the respective solvents must be of a type which does not interfere with the polymerization reaction between the two or more complementary monomers used; and 3) a solvent of a polarity distinct from that used in the continuous phase in the first stage reaction must be present. This solvent must be miscible with the continuous phase so that its polar nature can be significantly varied.

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The criteria for selecting a polymer system for use in the process of the invention are the following: 1) One of the two monomers must be hydrophilic and its complementary monomer must be hydrophobic; 2) the two monomers must react spontaneously upon contact to form polymer chains which are insoluble in both phases; and 3) reaction between the selected monomers should be as little inhibited as possible in the presence of the solvents used in the respective phases of the reaction system.

10 With regard to point 3, it should be noted that a certain degree of solvent interference, viz. hydrolysis side reactions are inevitable.

However, it is an important aspect of the process of the invention that a certain hydrolysis of the hydrophobic monomer can be tolerated without seriously affecting the quality of the membrane. The 15 local concentration of hydrolyzed monomer can be reduced to a minimum by the portion addition of monomer to the continuous phase.

Poly condensation reactions are suitable for the process of the invention, but polyaddition reactions can also be used. By careful selection of solvents selected in accordance with the teachings of this specification to suit the particular polymer systems and materials to be encapsulated, those skilled in the art will be able to produce capsule membranes of e.g. polyester from a polyol and a diacid halide, other 25 polyamides from diamines and diacid halides, polyurea from diamines and diisocyanates, and polysulfonamide from a difunctional sulfonyl halide and a diamine. Encapsulation processes using other polyaddition reactions, e.g. of the type disclosed in U.S. Pat. No. 3,864,275 is also within the scope of the present invention.

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The process according to the invention is further illustrated by the following examples:

Example 1.

1.5 ml of an aqueous carrier solution comprising polyvinylpyrrolidone,

Mix 5 albumin and 250 μΙ antisera for thyroxine with 50 µl of a 0.5M

tetraethylene pentamine carbonate solution CpH value 8.2 - 8.6 buffer with CO2). The aqueous phase is then added to 15 ml of cyclohexane containing 3-6% "ARLACEL" (sorbitanoleate) as an emulsifier. The 2-phase system is emulsified by means of a magnetic stir bar, and during the stirring, a 2 ml portion of a 4: 1 v / v solution containing 0.1 mg / ml of terephthahyl chloride is added to initiate the polymerization. .

60 seconds later, an additional 0.8 ml of the terephthaloyl chloride solution is added. After 60 seconds, 0.5 ml of pure chloroform is added to increase the affinity of the continuous phase to the poly-functional amines. Then, at 30 second intervals, a further 3 0.5 ml portions of pure chloroform are added.

After a total reaction time of 4 minutes, the emulsion is gently centrifuged and the supernatant is discarded. The microcapsules are washed 20 with pure cyclohexane and a 50% aqueous "TWEEN" ®-20 solution (sorbitan monolaurate) buffered to neutral pH with 0.3M NaHCO 3.

The above procedure results in capsules having a sufficient permeability to allow passage of thyroxine (molecular weight 777 Dalton) and lower molecular weight materials, yet is insufficient to allow antibody leakage from the interior of the capsules.

Example 2.

2.5 ml of an aqueous carrier solution containing polyvinylpyrrolidone, albumin, IsC04 / NaHCO3 buffer and 0.3 ml glucose oxidase are mixed with 1.2 ml 2.5M hexanediamine carbonate (pH 8.4 - 8.6). This aqueous phase is then added to 30 ml of a mixed solvent consisting of 50 parts cyclohexane, 5 parts chloroform and 3-5% sorbitanoleate as emulsifier. The 2-phase system is emulsified by means of an emulsifying tube probe.

With stirring, 2.6 ml of terephthaloyl chloride solution from Example 1 is added to initiate the polymerization. An additional 0.8 ml aliquot of the terephthaloyl chloride solution is added 30 seconds later. This addition is followed by the addition of 4 x 5.0 ml cyclohexane at 30 second intervals.

When a total of 3.5 minutes of polymerization reaction time has elapsed, the reaction is terminated and the microcapsules are "harvested" as indicated in Example 1. Glucose oxidase is retained inside the capsules while glucose (molecular weight = 180) diffuses through the membranes.

Example 3

A 4.0 ml aqueous phase containing 1.25M hexanediamine carbonate and lactate dehydrogenase is emulsified in 20 ml of pure cyclohexane containing 2% nonionic surfactant ("Arlecel"). With vigorous stirring, membrane formation is initiated as toluene diisocyanate is added to the emulsion. A total of 75 μ ml of diisocyanate is added by means of an infusion pump over 8 1/2 minutes in the form of a 5.0 ml aliquot of a solution consisting of 90% cyclohexane / 10% chloroform. Thus, the affinity of the continuous phase for the diamine is continuously increased until all the cyclohexane-soluble diisocyanate is added. The system is then stirred for a further 20 minutes. Two minutes prior to insulation of the caps, the membrane surface tackiness is reduced by the addition of 0.6 ml of 10% terepthaloyl chloride. These capsules are permeable to substances in the molecular weight range below approx. 1000 Dalton.

Claims (12)

Example 4. A hexanediamine carbonate solution (pH = 8.5 ± 0.1) is prepared by mixing 17.7mL of 1,6-hexanediamine and 32ml of water and bubbling CC 1 hour, or until the pH level is reached. Tereph-5 thaloyl chloride solution is prepared by adding 20 g of terephthaloyl chloride to 200 ml of organic solvent consisting of 4 parts cyclohexane and 1 part chloroform. Terephthaloyl chloride is dissolved by vigorous stirring and then the solution is centrifuged for 10 minutes at 2600 rpm. Any precipitate is discarded. Mix 10 750 ml of cyclohexane with 125 ml of "SPAN" ®-85 in a 2-liter mixer equipped with magnetic stir bar. With stirring, a mixed solution made from 25 ml of 15% polyvinylpyrrolidone - 4% bovine serum albumin, 40 ml of phosphate-buffered saline, previously mixed with 5 ml of antiserum, and 30 ml of hexanediamine carbonate solution, is added to the cyclohexane. When drops of the desired size are formed, 70 ml of terephthaloyl chloride are added. 30 seconds later, 37.5 ml of terephthaloyl chloride is added. 60 seconds thereafter, 25 ml of chloroform is added and at 30 second intervals an additional 3 25 ml of aliquots of chloroform are added. The microcapsules are isolated by centrifugation of the 2-phase reaction system, decanting the above liquid and mixing the capsules with "TWEEN" ®-20 (buffered with NaHCO 3) and phosphate buffered saline. The capsules contain polyvinylpyrrolidone and bovine serum albumin as a filler. Substances with a molecular weight of over 20,000 Daltons 25 (like most antibodies) cannot penetrate the membrane. Substances with a molecular weight below approx. 5,000 Daltons penetrate the membranes. A process for preparing microcapsules comprising membranes having an upper permeability limit within a selected range, characterized by performing the following steps: A) forming a 2-phase system comprising a hydrophobic, continuous phase and discontinuous phase of separated aqueous droplets containing a first hydrophilic monomer capable of forming a polymer upon reaction with a second, complementary hydrophobic monomer; B) dissolving a portion of said second monomer in the continuous phase to induce interface polymerization around the drops in the discontinuous phase; The method according to claim 1, characterized in that the continuous phase of the 2-phase system in step A) has low affinity for the first monomer such that a thin membrane is produced in step B) and in step C). for example, the affinity of the continuous phase is increased to the first monomer and an additional polymer layer is formed around the drops in the discontinuous phase. Process according to claim 2, characterized in that the affinity of the continuous phase to the first monomer is increased by dilution of the continuous phase with a polar solvent. Process according to claim 3, characterized in that the polar solvent is added portionwise during the polymerization reaction. Process according to claim 1, characterized in that in the 2-phase system of step A) the continuous phase is selected in such a way that it has a relatively high affinity for the first monomer, so that in step B) membranes 151212 comprising a thick polymer network, and in step C) the affinity is reduced in the continuous phase of the first monomer so that further polymerization occurs preferably within the polymer network. Process according to Claim 1, 5, characterized in that a substance which is unable to pass through the membranes produced in step D) is included in the aqueous drops of step A) as a filler material. Process according to claim 6, characterized in that the filler material is selected from the group 10 consisting of polyvinylpyrrolidone, polyethylene glycol, polysaccharides and albumin. Process according to claim 1, characterized in that the first monomer is selected from the group consisting of polyfunctional alcohols and amines and the second 15 monomer is selected from the group consisting of diacid halides, diisocyanates and difunctional sulfonyl halides. Process according to claim 1, characterized in that the first monomer is selected from the group consisting of 1,6-hexanediamine, tetraethylene pentamine and mixtures thereof. Process according to claim 9, characterized in that the second monomer is selected from the group consisting of terephthaloyl chloride, sebacyl chloride and mixtures thereof. C) changing the affinity of the continuous phase to the first monomer by changing the polarity of the continuous phase by dilution with a solvent of a second polar character; D) further polymerization is allowed to occur at the interface of the changed continuous phase; E) completion of the interface polymerization when microcapsules of the selected permeability have been prepared. Process according to Claim 1, 25, characterized in that the first monomer is a polyfunctional amine and the second monomer is a diacid halide. Process according to claim 1, characterized in that the second monomer is added portionwise during the polymerization reaction.