IE44127B1 - Encapsulation of labile biogical material - Google Patents

Description

This invention relates to a process for encapsulating chemically active substances, and particularly to encapsulating labile biological substances such as enzymes and hemoglobin, in a semipermeable membrane which will retain the encapsulated material, yet will freely allow smaller molecules to pass through the membrane to react with the encapsulated material and reaction products to pass out. Xt also relates to a method of controlling the size of the pores in the semipermeable membranes of the capsules.

Microencapsulation technology has grown rapidly in recent years and has found many applications. Attemps have been made, with varying degrees of success, to encapsulate biologically active substances such as hemoglobin, carbonic anhydrase, urease, asparginase, lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), chemically active materials such as ion exchange resins and activated charcoal, and various other enzymes and chemically active substances. Microcapsules of this nature have already proven to have great utility in, for example, artificial kidneys and fixed enzyme system, and show great promise in a number of other areas. As an example, U.S. Patent No. 3,522,345 to Thomas M. S. Chang discloses nonthrombogenic microcapsules which can be utilized, when fabricated about enzymes or detoxicants and placed in an extracorporeal shunt, to introduce e.g. oxygen, medicaments, 412 7 and enzymatic substances into the blood stream at controlled rates.

Xn Artificial Cells, by Thomas M. S. Chang, (Charles C. Thomas, Publisher; Springfield, Ill. 1972) a process for encapsulating biologically active materials in a semipermeable membrane is disclosed. The technique involves dissoluing the material to be encapsulated and a monomer in water, and forming an emulsion with the water as the discontinuous phase. When a second monomer capable of polymerization with the first is dissolved in the continuous phase of the emulsion, a polymerization occurs at the interface of the two phases, and a membrane is formed around the typically colloidal sized droplets of solution.

This procedure has drawbacks and limitations which curtail its potentially wide applicability and commercial success. Since many biologically active substances, e.g. enzymes, are extremely labile, the relatively harsh reaction conditions necessary for polymerization often result in a very low yield of operable encapsulated material. Encapsulation of even relatively hardy enzymes, such as urease, is characterized by yields at best between 35 and 40 percent. At one or more steps in the process, much of the encapsulated material is denatured.

U.S. Patent No. 3,522,346 (Τ. M. S. Chang) relates to microcapsules of controlled permeability. The process therein disclosed involves encapsulating aqueous compositions in membranes of controlled size, thickness, and permeability. Droplets of an aqueous composition are dispersed in an organic liquid medium immiscible therewith, and a component soluble in the organic liquid medium and capable of reacting with a component of the hydrophilic composition is added to the dispersion thereby produced to form a macromolecular membrane by interfacial coascervation, polymerization, or condensation at the surface of the droplets. Typically, a polymer, condensation product, or a 4137 - 4 component thereof is added to the organic liquid and the membrane is formed about the droplets by interaction of the polymer, condensation product or component with a component of.the dispersed droplets, which may be a precipitant, a condensation or polymerization catalyst, or a compound of the eventual condensation product. The effective pore size of the encapsulating membrane is a function not only of the membrane composition but also of the thickness of the membrane and is selected in accordance with the intended use of the capsules. By suitable choice of operating conditions, pore size may be varied, but the reaction conditions necessary to ensure sufficiently strong yet selectively permeable membranes are in any case difficult to control and in some cases unknown. Also, the procedure produces capsules generally having membranes with erratic uniformity and porosity, many of which are useless for their intended purpose.

According to the present invention, there is provided a polymerization process for encapsulating a chemically active core material in a semipermeable membrane synthesized by an interfacial polymerization reaction, said process including the steps of: dissolving said core material in a first solvent chosen from the group consisting of water, hydrophilic solvents, and mixtures thereof to form a first solution: dissolving a first monomer in said first solution; adding a hydrophobic solvent to said first solution, said first monomer being less soluble in said hydrophobic solvent, than in the first solvent; emulsifying the two phase system thereby produced, said first solution forming a discontinuous droplet phase in the emulsion; adding a second monomer to said emulsion in fractions at intervals over the duration of the polymerization,said second monomer being capable of forming a polymer with 412 7 - 5 said first monomer and being highly soluble in said hydrophobic solvent, to form a highly permeable, macroporous capsule membrane about the discontinuous phase droplets; stopping the polymerization reaction by separating the macroporous capsules from said hydrophobic solvent; resuspending the capsules in a liquid in which said second monomer is highly soluble and in which said first monomer is less soluble than in the hydrophobic solvent; adding a second portion of said second monomer to the resuspended capsules in the said liquid, to cause a second polymerization to occur at said capsule membranes, to strengthen them and to render them impermeable to said core material; and quenching said polymerization.

In general, the invention is characterized by a twostep polymerization process using at least two monomers which will polymerize, one of which is soluble in a hydrophobic solvent, the other of which is soluble in a hydrophilic solvent or water. First, a hydrophilic solution is prepared, preferably with water, by dissolving the material to be encapsulated in the water together with a first monomer. Since many monomer solutions have a pH which would denature many labile biological substances, the monomer solution often must be buffered to a pH range between 5 and 9 before labile biological material is added.

A hydrophobic solvent is then prepared which has the following properties. First, the solubility of the second monomer which is to be used in the polymerization should be very high in this hydrophobic solvent. Second, the hydrophobic solvent should have a slight affinity for the first monomer. Third, the hydrophobic solvent should be such that the hydrophilic solvent, when added to it, will form a good emulsion.

The two solvents are then added together and emulsified, the hydrophilic solvent, typically, being the discontin - 6 uous phase. Emulsification can be accomplished by any one of several well-known techniques, usually with the aid of an emulsifying agent. The size of the droplets produced dictates the size of the microoapsules formed.

When the desired droplet size range has been achieved, the second monomer is added to the emulsion in increments and polymerization, condensation, or polyaddition, occurs at the interface of the two-phased system. Because the first monomer, dissolved primarily in the discontinuous phase, is slightly soluble in the continuous hydrophobic organic phase, some diffusion occurs into the continuous phase. The microcapsule membrane forms across this interface zone as the polymerization proceeds. The forming membrane limits the further diffusion of the first monomer into the continuous phase, with the membrane characteristic very dependent on the detailed sequence of random encounters between the monomers.

Generally, the membranes produced to this point are macroporous with successful semipermeable membranes occurring only under exceptional conditions which are difficult to control. It is believed that the problem results from the presence of defects in the membranes which result in macroporous holes through the membranes after the reaction is quenched and the capsules are washed. In the prior art, this is overcome only by continuing the polymerization, condensation, or polyaddition for long'enough to close these large defects, which simultaneously closes the smaller pores. Acceptable capsules thus are believed to result only over a narrow range of polymer formation in which the defects are closed and the smaller pores are still open.

However, it has been discovered that if such microcapsules are separated from the continuous phase, resuspended in a quantity of different hydrophobic solvent in which the solubility of the first monomer is greatly reduced, and then exposed again to the second monomer, the 412 7 same polymerization reaction occurs at a much slower rate over a much narrower interface zone lying only within the interstices of the capsule membrane. This results in the patching of the macroporous defects in the capsule's wall, decreasing the pore size, and strengthening the membranes.

It is believed that this second polymerization reaction bridges selectively more across the larger pores where the first monomer diffuses closer to the continuous phase, and that this patching proceeds substantially without significant increase in the capsule wall thickness. By contrast, the continued use of the first solvent increases the wall thickness, decreasing the permeability of most of the membrane for the sake of sealing the defects.

It has been found that this secondary polymerization technique is useable with a large number of known encapsulation processes to form semipermeable capsules and to control the pore sizes thereof.

With this two-step polymerization process, it is possible to choose the second monomer for use in the continuous phase which, in conventional procedures, would quickly denature labile biological materials sought to be encapsulated. The technique for avoiding such denaturing involves controlling the relative concentration of solutes in the continuous and discontinuous phase so that the concentration of the second monomer in the continuous phase is kept to less than optimal values, and the solutes in the discontinuous phase are in high concentration. In this situation, diffusion of the second monomer into the discontinuous phase is inhibited and denaturing of the labile biological material is minimized, but a macroporous, poorly formed,and clearly unsatisfactory capsule membrane is produced. However, in the second polymerization step, the concentration of the second monomer in the continuous phase of the resuspension may be safely increased (since the encapsulated material is now protected by the raw capsule - 8 membrane) to strengthen the capsule membranes and render them semipermeable.

The second monomer is added to the emulsion in the first polymerization step, not all at once to initiate poly5 merization, but rather in fractions, at regular time intervals, over the duration of the polymerization. This technique maintains the concentration of the second monomer at the interface at a more constant optimum level at all times in the formation of the first coarse membrane and thus inhibits diffusion of the second monomer into the discontinuous phase and increases the yield of operable labile biological material. This technique is also quite useful for systems in which hydrolysis of the chosen second monomer is a competing reaction with the polymerization.

Xn one important embodiment, the first monomer is a diamino monomer and the second monomer is a dibasic organic acid halide; the initial hydrophobic solvent is a solution comprising 80% by volume cyclohexane and 20% by volume chloroform; and in the resuspension, the hydrophobic liquid is pure cyclohexane. The diamino monomer is essentially insoluble in cyclohexane, and slightly soluble in chloroform.

When encapsulating hemoglobin, it has been discovered that'if the red blood cells are dehydrated by exposure to hypertonic saline solution, a hemoglobin solution with concentrations of up to 30 g/dl may be prepared, and the high salt concentration inhibits diffusion of water molecules into the discontinuous phase where they react with molecules of the second monomer, rendering them inoperable.

Since it is well known that many labile biological materials rapidly denature in highly acidic or basic environ ments, at all stages of the novel encapsulation process, the pH of the immediate environment of the material sought to be encapsulated should be kept at least within the range of 5 to 9. When a diamino compound is used as a first monomer, this is accomplished by bubbling C02 through the 412 7 - 9 diamino solution before it is added to the labile biological material. The diamino solution, which typically has a pH on the order of 11.5, is converted to diamino carbonate with a typical pH of about 8.6-8.7. If saturated with COg and stored beneath 100% COg gas, this solution can be brought to a pH of about 8.4. In some cases, a polyamine will be added to the diamino monomer before the hydrophobic solvent is introduced. The pH of the polyamine, e.g. tetraethylenepentamine, may be adjusted to between 5 and 9 before addition to the said monomer.

By contrast, the prior art utilized the weak NaHCOgNagCOg buffer system, i.e., the salts of a strong base and the weak acid, where the present invention has used the weak acid itself. The resulting pH of the prior art, buffered diamino-hemolysate solution is pH 11.

A principal advantage of the present technique for the immobilization of biologically and/or chemically active materials to form solid or pseudo-solid reactors, is that the materials are encapsulated In bulk without direct competition with impurities for available binding sites.

Thus highly active capsules can be made from relatively poorly purified materials. In addition, the active materials are protected from attack by microorganisms often responsible for the inactivation or inhibition of such reactor components.

A first aspect of this invention utilizes a variation in the well-known process for microencapsulation known generally as interfacial polymerization. Two mutually immiscible solvents are chosen, one being hydrophobic, the other being hydrophilic or, preferably, water. The material to be encapsulated and a first monomer are dissolved in water, the two solvents are mixed, and then the mix is emulsified, the hydrophobic solvent being the continuous phase. The size of the colloidal droplets determines the size of the microcapsules to be produced. Emulsification may be 127 accomplished by any of the well known emulsification techniques such as, for example, using a blender, and is usually done with the addition of an emulsifying agent. Since the size of the droplets produced in any given technique will vary within a specific range, a filter may be used to separate over-sized capsules made in any given run to minimize differences in capsule diameter. For a more detailed disclosure of the method for varying capsule size, reference should be made to Thomas M.S, Chang, Artificial Cells, chapter 2.

When droplets of sufficient size have been produced, a second monomer capable of forming a polymer with the first monomer by polycondensation or polyaddition is introduced into the suspension. Polymerization occurs only at the interface of the two-phase system. Obviously, the monomer must be chosen from among those which exhibit suitable solubility properties in the solvents chosen.

To maximize yield of operable, i.e. active, biological material enclosed within the capsules, reaction conditions must be carefully controlled so that the generally sensitive material will not denature to any significant extent during the polymerization. Accordingly, it has been discovered that a most critical factor in obtaining a high yield of operable encapsulated material is the pH of the discontinuous phase. To ensure against denaturing of labile material, the pH must be controlled at all times during the process, generally within the range of 5 and 9. The solvents must be chosen, especially the hydrophilic one, from among those which will not quickly denature the material to be encapsulated. The choice will be well within the ability of men skilled in the art after they have read the disclosure. To facilitate proper dispersion and separation, the two solvents should have specific gravities slightly different from each other.

Polymerization which occurs in a pH environment which 412 7 - 11 is hostile to the particular chemically active substance sought to be encapsulated should not be used for that substance. However, many monomers, which in solution have a natural pH capable of denaturing most labile biological material, may be buffered to within the above disclosed pH range without affecting their activity. Other monomers can be used without buffering, depending on their natural pH and the nature of the material sought to be encapsulated.

For highly alkaline monomers, passing C02 gas through the solution is a preferred method of bringing the pH within the nondenaturing range.

Xt has been discovered that a much stronger capsule membrane having fewer imperfections and using a minimal degree of polymerization can be produced by a two-step polymerization. In this regard, it has been discovered that the capsule membranes produced by the above-described single step process are often macroporous, uneven, and vary considerably in strength and thickness. However, if the polymerization at the interface of the emulsified system is quenched by removal of the continuous phase, and the capsules are then resuspended in fresh solvent which has a lower solubility for the first monomer than the hydrophobic solvent which formed the continuous phase, an additional portion of the second monomer added to the resuspension will cause further polymerization which will strengthen the membranes, cause the larger pores of the membranes to fill in preferentially, and thereby avoid the macroporous condition. The process hence produces a high quality, semipermeable membrane by the use of a second, minimal amount of polymerization, the latter being used to maximum additional advantage to fill in the membrane imperfections.

Further refinements of this two-step polymerization process enable control of the strength, thickness and porosity of the membrane to an improved degree. If the - 12 hydrophobic solvent comprising the continuous phase of the emulsion is chosen such that the first monomer is slightly soluble therein, monomer from the droplets diffuses slightly into the continuous phase. This results in a relatively thick phase boundary and an equally thick and usually porous membrane, formed wherever the two monomers come into contact. Generally, the greater the solubility of the first monomer in the continuous phase, the more macroporous the membrane produced. If, on the other hand, the first monomer is much less soluble in the contin uous, hydrophobic phase, the boundary between the phases is sharp, and a thin, dense, and usually microporous membrane results. When the partly formed capsules are resuspended, a different solvent is used having a different solubility for the first monomer than the solvent used as the discontinuous phase in the first polymerization. When a macroporous membrane has been produced in the first polymerization, a solvent in which the first monomer is essentially insoluble is used in the second polymerization and has the effect of hardening the membranes. It is believed that in the second polymerization step, the two precursors to the polymer meet only within the weak spots of the imperfect membrane produced up to that point, i.e., where there is preferential diffusion of the first monomer towards the solvent. Thus, more polymer is formed to fill in the larger pores.

The concentrations of the monomers in their respective solvents can be controlled to further advantage. If, for example, the second monomer, dissolved in the hydro30 phobic, continuous phase of the emulsion, has the property of denaturing the material sought to be encapsulated, the first monomer, dissolved in the discontinuous phase, may be introduced at a high concentration relative to the second, of the order of 100 to 1, to prevent any migration of the denaturating monomer into the droplets of solution 412 7 - 13 during the first polymerization. This technique often produces a highly porous microcapsule, but, in the second polymerization step, the concentration of the second monomer may be safely increased and if a proper solvent is used, the capsules will be strengthened and rendered microporous by selectively bridging across the larger pores with a thinner membrane, as explained above, without seriously affecting the encapsulated material.

When encapsulating hemoglobin, it has been found that the concentration of hemoglobin encapsulated can be increased significantly if red cells used to make hemoglobin hemolysate are throughly washed in hypertonic saline. In addition, the relatively high salt concentration in the aqueous, discontinuous phase of the emulsion aids the interfacial polymerization of the first monomer by limiting the solubility of water in the organic phase containing the second monomer, which, typically, is rapidly decomposed on exposure to water. It is believed that the reaction products of such hydrolysis of the second monomer contribute to the imperfections of the membrane if not removed in the separation step.

The invention will be further clarified and understood by the following examples.

Example 1.

Red cells from expired blood bank whole human blood were separated and then washed two times in twice their volume of hypertonic saline (1.5 g/dl) by centrifuging in a refrigerated centrifuge at 2800 xg for five minutes. Hypertonic saline was used to remove water from the cells in order to produce the highest possible concentration of hemoglobin after the lysis of the cells. A third centrifugation for 15 minutes at 2800 xg served to pack the cells. As much saline as possible was removed from the packed cells which were then lysed by shaking vigorously with one ml anhydrous ether per 10 ml packed cells. This mixture was - 14 centrifuged at 1000 xg for 20 minutes and the cell debris and ether which rise to the top removed. Hemoglobin concentration was checked by measuring the absorbance of 10 μΐ of the hemolysate in 4.0 ml of isotonic saline at a wavelength of 540 nm. The absorbance readings from all properly prepared hemolysate solution were between .7 and .8, which correspond to a concentration of 28 to 32 g/dl. Hemoglobin concentrations greater than 30 g/dl crystallized and precipitated on storage at refrigerator temperature (4°C). The hemolysate could be used for at least a week if refrigerated.

Hexanediamine carbonate solution was prepared by bubbling carbon dioxide into a 3M solution of 1.6 hexanediamine (Eastman Kodak Co.) until the pH reached 8.5. This required about 15 grams of C02 per 100 ml of solution.

Ten milliliters of hemolysate solution containing 30 g/dl hemoglobin was mixed with 10 ml of 3M hexanediamine carbonate solution. This aqueous solution was then added to 125 ml of pre-chilled hydrophobic organic liquid made from 100 ml of 20% chloroform-80% cyclohexane mixture and 25 ml p of an emulsifying agent, SPAH—85 (Sorbitan trioleate).

Emulsification was then started in a pre-chilled blender and continued for 1 to 2 minutes.

Terephthaloyl chloride, the second monomer, required preliminary purification. Hot (nearly boiling) cyclohexane was saturated with terephthaloyl chloride (Practical gradeEastman Kodak Co.) and the solution quickly filtered. The clear liquid was collected and allowed to cool and crystallize in a closed container. The crystals were collected with as little exposure as possible to the atmosphere and dried in a vacuum as this compound reacts with atmospheric moisture. The stock solution of terephthaloyl chloride was prepared by dissolving 10.15 grams of purified terephthaloyl chloride in 95 ml of 20% CHCl3-8O% cgHi2' v/v* A ,5M solution resulted. This solution should be kept in a tightly 412 7 sealed container and may be used for at least a month or until precipitate begins to form.

Three ml of the 5M terephthaloyl chloride in 20% chloroform-80% cyclohexane organic solution were then added to the emulsion while blending at a low speed. The acid halide is added in equal increments during polymerization at 30 second intervals. The final concentration ratio of diamino/dibasic organic acid halide was of the order of 100 to l,in fact 128 to 1, although some variation is tolerable, 1.0 At the end of three minutes, the first polymerization reaction was complete and the suspension was centrifuged for 15 to 20 seconds at 400 to 500 xg. The supernatant organic liquid was then removed by aspiration.

The capsules were then resuspended in a second liquid consisting of 100 ml cyclohexane and 10 ml sorbitan trioleate. While mixing slowly, 7 ml of .5M terephthaloyl chloride in 20% chloroform and 80% cyclohexane organic solution were added in equal increments at 30 second intervals. This hydrophobic liquid contains only 1.0 to 1.5% chloroform versus 20% in the first organic stage. The reduced chloroform content cuts down the solubility of diamino monomer in the solvent making a thinner membrane and patching holes in the original coarser one. The diamino/dibasic organic acid halide concentration ratio in the second liquid was less than 60 to 1, in fact 50 to 1. Stirring was continued for 3 minutes at which time the reaction was quenched by the addition of 30 ml of 50% TWEEN-20 (sorbitan monolaurate) buffered to neutral pH with .3M NaHCOg. TWEEN is a Registered Trade Mark.

Note that the hemoglobin-enzymes hemolysate solution was at no time exposed to a pH less than 5 or greater than 9. Hexanediamine solution has a natural pH of 11.5 and a pH of about 11 when mixed with hemoglobin. Bubbling the C02 therethrough causes the production of H2C03 and hexane35 diamine carbonate. The C02 acts as a buffer and is preferred over sodium salts or weak acids. Before buffering, the sorbitan monolaurate used had a typical pH of approximately 4.5.Extended exposure to this acidic environment would denature the hemoglobin and enzymes. This can be avoided by buffering a hypertonic solution of the sorbitan monolaurate to have a pH close to 7. The purpose of the sorbitan monolaurate is to facilitate transfer of the microcapsules into an aqueous medium such as saline, (see Artificial Cells, chapter 2). Xt should be noted that in this step sorbitan monolaurate tends to react slowly with the terephthaloyl chloride to produce a sticky polymer coating and, in addition, to crenate the microcapsules. A superior alternative procedure is to remove as much of the solvent as possible by centrifugation and then follow through with the above TWEEN-20 addition and subsequent separation.

Microcapsules produced by this process did not allow leakage of enzymes or hemoglobin. Half equilibrium times (t%) for solutes of various molecular weights are given in TABLE X. Half equilibrium time (t%) is an indirect measure of the permeability of the wall of a microcapsule to a given solute, and is one half the time taken for attainment of equilibrium between the rates at which molecules of said solute traverse the wall into and out of a microcapsule immersed in a solution of said solute. 413 7 TABLE I PERMEABILITY OP MICROCAPSULES - HALF EQUILIBRIUM TIMES Substance Molecular Weight t 1/2 + SEM (sec.) Glycerol 92 1.75 + .1 Diacetone Alcohol 116 2.5 + .2 Glucose 180 4.9 + .1 Sucrose 342 10.6 + .2 Tris a 121 2.8 + .1 „. . b Bicine 163 6-1 + -3 CAPS C 221 8.0 + .5 n · 4 Pipes 302 13.0 + .5 Tris (hydroxymethyl) aminomethane, pH 8 b Ν,Ν-bix (2-Hydroxyethyl) glycine, pH 8 c Cyclohexylaminopropane sulfonic acid, pH 8 c J 3 Piperazine-N-N' -bis (2-ethane sulfonic acid), pH 8 The activity of four enzymes, lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), urease, and p-glucuronidase was measured before and after encapsulation. LDH and GOT were found naturally in the red cell hemolysate. Urease and β-glucuronidase were added to hemolysate in the form of lyophilized partially purified enzyme, available from commercial sources. The results of the comparisons for LDH, GOT, and urease are disclosed in TABLE II. Activity was measured in international units (IU) per liter of sample under conditions of the test reaction. The yields determined for β-glucuronidase were uniformly above 50¾. 4412Y TABLE II Enzyme Activity (IU/1) + SEM Retained activity a (%) LDH 4120 + 90 55 GOT 1200 + 80 66 Urease 234000 + 8000 68 a The enzyme activity of the microcapsules divided by the enzyme activity of the hemolysate used in preparing the microcapsules, (% yield) (SEM=standard error for the mean) TABLE III shows a comparison of the present saturation of oxygen in hemolysate solution used to prepare the microcapsules with encapsulated hemoglobin, at various 02 press5 ures.

TABLE III Pressure 02 Percent saturation Hemolysate hemoglobin Encapsulated hemoglobin 5 7% 5% 10 . 21% 10% 15 46% 20% 20 62% 32% 25 74% 45% 30 82% 58% 35 89% 68% 40 92% 74% 50 97% 83% 60 99% 89% 70 99%+ 92% 80 99%+ 94% 90 99%+ 96% 412 7 - 19 TABLE III (continued) Pressure 0% Percent saturation Hemolysate hemoglobin Encapsulated hemoglobin 100 99%+ 97% 110 99%+ 98% 120 99%+ 99% Table IV shows the effect of varying the amount of the acid halide in the discontinuous phase of each polymerization on the porosity of microcapsules prepared in one series of tests, the porosity being measured indirectly by observing whether or not the encapsulated material leaked through the membrane. As the table clearly indicates, the concentration of the second monomer in each polymerization step is highly material to good capsule formation. Those skilled in the art will have little difficulty determining the optimal concentration ratio to be used with other polymer systems. The capsules of Table IV were made with the procedure outlined above, except that emulsification was performed in SPAN 85 alone before the mixed organic solvent was added. Results of other variants of the procedure are qualitatively similar. - 20 TABLE IV MICROCAPSULES MADE WITH 10 ML OF HEMOLYSATE AND VARIOUS AMOUNTS OF .5M TEREPHTHALOYL CHLORIDE First Reaction Second Reaction Capsule Condition Terephthaloyl Chloride Terephthaloyl Chloride 1 2 ml 4 ml Very leaky 2 2 ml 8 ml Very leaky 3 3 ml 7 ml No leak 4 3 ml 7 ml No leak 5 4 ml 7 ml No leak 6 4.5 ml 7.5 ml Mod. leaky 7 4.5 ml 7.5 ml Mod.leaky 8 5 ml 8 ml Very leaky 9 5.5 ml 8 ml Very leaky Microcapsules separated from the organic solvent after the first reaction were red, indicating that little or no denaturing had occurred, but were found to be poorly formed and leaky. Increasing the terephthaloyl chloride concentration under these conditions produced microcapsules which were brown, indicating leakage and hemoglobin denaturation. Increasing the reaction time of the first reaction up to five minutes had little apparent effect on the microeapsules, but increasing the second reaction time to more than four minutes caused the microcapsules to turn brown, indicating hemoglobin denaturation. Decreasing either reaction time one minute or more resulted in leaky microcapsules. The presence of small amounts of ethanol commonly used as a stabilizer for the chloroform can necessitate the readjustment of optimum volumes. To achieve the greatest repeatability and most reliable capsule formation, the 412 7 - 21 ~ ethanol should be removed. The chloroform used in Table IV contained some ethanol. With the ethanol extracted the optimum second reaction volume is typically 3-4 ml rather than 7. Decreasing the amount of TWEEN-20 used in the separation step resulted in poor separation of the microcapsules from the organic solvent. Microcapsules which were allowed to remain in TWEEN-20 for excessive times (>): hour) turned brown. Microcapsules stored in unbuffered saline which has a pH of 5.5 turned brown after several days, while those stored in a solution buffered to pH 7.4 remained red for months.

Further study of the capsules made in this example indicate that they have a ratio of wall volume to capsule volume on the order of .028. When t is the wall thickness and D is the overall diameter, the ratio of wall volume to capsule volume is given by: vwall irD2 t R = - , - = 6 t/D vcapsule it/6 D3 The capsules had an average wall thickness (t) of 700A or .07 u and an average diameter (D) of 15 μ. Hence Rib X .07/15 or .028. Given that nylon has a density approximately equal to 1, 28 mg of nylon would be necessary to form the solid membranes per ml of capsules produced. The total amount of diamino constituent present, as can be calculated from the concentration used, is 1.5 mM/ml or 174 mg/ml.

Since the nylon wall is 46% by weight diamino constituent, 12.9 mg of diamino constituent, or about 7.5% of the total would be necessary to form a solid nylon wall. Since, however, the acid chloride is the reaction limiting constituent and the ratio of diamino/dibasic organic acid chloride was at best 50/1, the upper limit of nylon produced is on 44137 the order of 2.0% of the total diamino constituent. This indicates that the membranes produced are most likely sponge like with an open cell structure.

These capsules were impermeable to hemoglobin (mw = 68,000) but permeable to insulin (mw = 12,000,half equilibrium time between 2 and 3 minutes). A comparison of the half time for insulin with those for low molecular weight substances such as those given in Table X indicates a nonlinear relationship. It is believed that this phenomenon is caused by the steric properties or charge state of the insulin molecules.

Example 2. 1.5 ml of bovine albumin (30%) is added to 1 ml of the DBDSA-hexanediamine carbonate monomeric solution of Example 1 together with 0.4 ml 36% KC1 and a polyamine consisting of 0.1 ml 2.9M tetraethylenepentamine (the polyamine was previously adjusted to pH 9.5 with HCl before adding to the diamino monomer-containing solution). 4.0 ml of SPAN 85 and 10 ml of the hexane-chloroform solvent of Example 1 is then added to the mix and an emulsion is produced, in 1-2 minutes, by vigorous stirring using a magnetic stirring bar. 0.4 ml. of terephthaloyl chloride solution (0.5M in hexane-chloroform solvent of Example 1) is added in 0.1 ml increments every 30 seconds, followed by addition25 al 0.1 additions every minute for the next 6 minutes.

After Centrifuging to separate the capsules, the supernatant is discarded, and the raw microcapsules are resuspended in cyclohexane containing 5% SPAN 85. 0.7 ml terephthaloyl chloride are added, and the suspension is stirred for 3 minutes. After separation of the phases by centrifugation, the microcapsules are washed once in pure cyclohexane and subjected to final washing and recovery as indicated in Example 1.

Labile biological materials, including enzymes, may 412 7 - 23 be dissolved in the albumin solution as desired. The tetraethylenepentamine helped to cross-link the polymer to form strong, uniform pore size semi-permeable membranes.

While the foregoing examples are limited to polycondensation systems which include a dibasic organic acid halide and a diamino monomer to produce a polyamide polymer or copolymer, it will be obvious to those skilled in the art that the two step polymerization process and other teachings herein disclosed will be useful in many other prior art encapsulating processes. By astute selection of solvents, chosen in accordance with the teachings of this invention to suit particular polymer systems and particular materials to be encapsulated, those skilled in the art will have little difficulty in producing high quality capsule membranes of, for example, polyester, from a polyol and a dibasic organic acid (HOOC-R-COOH) or dibasic acid halide (C10C-R-G0C1), other polyamides, from a diamine and a dibasic organic acid, polyurea, from a diamine and a diisocyanate, polycarbonate, from a diol and an acid halide, polysulfonamide from a multifunctional sulfonyl halide and a diamine, as well as cross-linked protein, polyphthalamide, and other well-known polycondensations.

Encapsulating procedure using polyaddition reactions such as the type disclosed in, for example, Kan et al.'s U.S. Patent No. 3,864,275, are also within the scope of this invention. Those skilled in the art will have little difficulty adapting the above-disclosed process to systems utilizing polyfunctional amines and, for example, epichlorohydrin or polyesters containing epoxy groups.

To summarise we have described hereinbefore high yield techniques capable of encapsulating in their active states many chemically active substances such as enzymes and other labile biological materials, wherein very strong semi-permeable encapsulating membranes are formed. Microcapsules have been obtained which exhibit superior uniformity of semi44137 - 24 permeability. Thus, their walls are uniform and without porous defects, and possess a pore size range such that the encapsulated material cannot escape from the capsules but can readily react with appropriate substances in the envir5 onment in which the capsules are placed. The conditions under which, and the polymer materials from which the encapsulating membranes are formed can be varied to suit the material to be encapsulated, and to control the pore size range thereby to render the membranes selectively permeable to molecules having dimensions smaller than the upper limit of the said pore size range. The techniques disclosed include encapsulating a high concentration of hemoglobin in a capsule permeable to oxygen, carbon dioxide and other small molecules.

Claims (7)

1. A process for encapsulating a chemically active core material in a semipermeable membrane syntesized by an interfacial polymerization reaction, which process comprises 5 dissolving said core material in a first solvent chosen from the group consisting of water, hydrophilic solvents, and mixtures thereof to form a first solution; dissolving a first monmer in said first solution;/' adding a hydrophobic solvent to said first solution 10 emulsifying the two phase system thereby produced; said first solution comprising a discontinuous droplet phase in the emulsion; and adding a second monomer to said emulsion, said second monomer being capable of forming a polymer with said first monomer and being 15 highly soluble in said hydrophobic solvent, to form a membrane about the droplets of said discontinuous phase, wherein the process additionally comprises adding said second monomer to said emulsion in fractions at intervals over the duration of the polymerization, 20 stopping the polymerization reaction, at a stage wherein a highly permeable macroporous capsule membrane has been formed, by separating said hydrophobic solvent from said emulsion; resuspending the capsules in a liquid, said second monomer being highly soluble in said liquid, said first monomer being less 25 soluble in said liquid than in said hydrophobic solvent; adding a second portion of said second monomer to the resuspension, to cause further polymerization at said capsule membranes, to strengthen them, and to render them impermeable to said core material, and quenching said polymerization.

2. The process as set forth in claim 1 wherein said first monomer is a polyamine.

3. The process as set forth in claim 2 wherein said second 5 monomer is a dibasic organic acid halide.

4. The process as set forth in claim 1 wherein said core material is a labile biological substance.

5. The process as set forth in claim 4 wherein said labile biological substance is an enzyme. 10

6. The process as set forth in claim 1 wherein the pH of the discontinuous phase is maintained between 5 and 9.

7. The process as set forth in claim 1 wherein said interfacial polymerization reaction is a polycondensation or a polyaddition reaction.