Note: Descriptions are shown in the official language in which they were submitted.
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
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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
--2--
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
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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
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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
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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
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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
--12--
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.
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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.
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