Note: Descriptions are shown in the official language in which they were submitted.
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WO 99/48480 PCT/EP99101626
Process for the production of microcapsules
Description
The present invention relates to an improved process for the production of
microcapsules.
Microcapsules are produced by encapsulation of finely dispersed liquid
phases by enveloping with film-forming polymers. Microcapsules are
especially used in the area of depot preparations, accordingly the active
compound contained in the microcapsules is protected by the shell of the
microcapsule and is not released immediately, but only with a delayed
release.
It is known to produce microcapsules by atomization of a polymer solution
and active compound by means of ultrasound, the liquid droplets produced
in this way being sprayed into a precipitation bath.
Thus US-A-4 352 883 describes a 2-stage process for the production of
microcapsules, in which living cells, such as, for example, islet of
Langerhans cells, are encapsulated. For this, the living cells are
suspended in sodium alginate and this suspension is sprayed into a
precipitation bath which contains polyvalent cations (for example Ca2+).
The physical crosslinking of the alginate on the surface occurs here due to
the polyvalent cation. In the second step, the capsules produced in this
way are mixed with a cationic polymer, which brings about further physical
crosslinking. Polycations mentioned in this publication are polyethylene-
imine and polylysine.
US-A-5 389 379 discloses a process for the production of microcapsules in
which the liquid droplets produced by means of an ultrasonic nozzle are
first introduced into a liquid in which the liquid droplets are not soluble
(for
example into ethanol). This liquid is then replaced by water. This
complicated 2-stage process is chosen because direct introduction of the
liquid droplets is not possible, since otherwise instead of the microcapsules
a thin polymer film forms on the surface of the water. The size of the
microcapsules formed in this way is specified as 10 to 1000 Vim.
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In US-A-5 472 648, a process for the production of microcapsules is
specified in which small liquid droplets are produced from an alginate
solution by means of ultrasound and are sprayed into a vessel with a
CaCl2 solution. After the liquid droplets have hardened in this CaCl2
solution (precipitation bath), the microcapsules obtained in this way are
brought out of the CaCl2 solution with the aid of a conveyor device (belt
sieve). In order to produce microcapsules which are as uniform as
possible, it is proposed in this publication to additionally add a surface-
active agent to the CaCl2 solution in order to lower the surface tension, or
to foam the CaCl2 solution in order to lower the mechanical stress on the
liquid droplets when impinging onto the CaCl2 solution. The size of the
microcapsules obtainable in this way is specified as 100 to 4000 Vim.
US-A-5 484 721 describes a process for the production of capsules
containing microorganisms. For this, the aqueous solution containing the
polyanion is atomized by means of compressed air and a spray nozzle and
the liquid droplets thus obtained are introduced into a liquid film which
contains calcium or potassium ions as crosslinkers. The size of the
microcapsules produced according to this publication is specified as
between 10 ~m and 4 mm. However, these microcapsules are not suitable
for the encapsulation of active compounds which are to be released in a
delayed manner.
US-A-5 589 370 relates to the production of microcapsules by salting out
polymers. The microparticles produced in this way, however, dissolve
immediately, as soon as they are added to water. To this extent, they are
not suitable for the production of depot preparations.
The object of the present invention consists in the provision of a process
for the production of microcapsules which are suitable for the production of
depot preparations having delayed release, and which can be produced in
such a size that they can also be used for the parenteral administration of
depot preparations.
This object is achieved by a process for the production of microcapsules by
atomization of an aqueous solution 1, which contains 0.1 to 5% by weight
of at least one water-soluble polyanion, to give liquid droplets, the liquid
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droplets thus obtained impinging onto a flowing film of an aqueous solution
2, containing:
~ 0.1 to 5% by weight of calcium cations; and
~ 0.001 to 0.4% by weight of chitosan having a number-average molecular
weight of greater than 40,000 g/mol; andlor
~ 0.1 to 5% by weight of chitosan having a number-average molecular
weight of between 500 and 40,000 g/mol.
The microcapsules produced according to the process according to the
invention have a particularly stable crosslinkage of the outer shell, which is
especially to be attributed to the simultaneous use of calcium cations and
polycations as crosslinking agents. On account of this property, these
microcapsules are particularly suitable for the encapsulation of active
substances for the production of depot preparations.
The process according to the invention additionally has the advantage that
no agglomeration or caking of the microcapsules occurs, as is the case, for
example, when the solution 1 atomized by means of ultrasound is sprayed
into a stirred precipitation bath of the solution 2.
The film can flows over a fixed substrate according to the process
according to the invention, the substrate preferably not being arranged
horizontally. According to a particularly preferred embodiment, the
substrate forms an inclined or a vertical plane.
It has proven advantageous if the water-soluble polyanion is an alginate, in
particular an alginate having a high guluronic acid content.
The water-soluble polyanion, however, can also be selected from the group
consisting of carrageenan, sulfated polysaccharides, gelatin and agar-agar.
According to a particularly preferred embodiment of the present invention,
the solution 1 additionally contains at least one polyacid or its alkali metal
salt, selected from the group consisting of polyamino acids, poly-
phosphates and polysulfates of polysaccharides.
Preferred examples of a polyphosphate are sodium polyphosphate and a
polyphosphate of a polysaccharide.
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The polysaccharide can be selected from the group consisting of starch
hydrolyzates, inulin, hydroxyethyl starch, xylan and dextrans.
As a polyamino acid, it is preferred to use polyaspartic acid or polyglutamic
acid.
According to a further advantageous embodiment of the invention, the
solution 2 additionally contains a polycation selected from the group
consisting of polylysine, polyvinylamine, poly-a,~i-(2-2dimethylaminoethyl~
D,L-aspartamide, aminated polysaccharides, such as, for example,
aminated dextrans, cyclodextrins, cellulose ethers, starches, pectins, and
their hydrophobically substituted derivatives.
The release can moreover be produced by reacting the microcapsules,
after preparation of the particles, in an additional process step with a
crosslinker selected from the group consisting of glyoxal, glutaraldehyde,
succinaldehyde, or dicarboxylic acids, such as, for example, oxalic acid,
succinic acid, fumaric acid, malefic acid, malic acid, glutaric acid, adipic
acid, 2,3-O-isopropylidenetartaric acid, diacid chlorides, such as, for
example, succinyl chloride, fumaryl chloride, glutaryl chloride, adipoyl
chloride, or tricarboxylic acids, such as, for example, citric acid, 1,2,3-
propanetricarboxylic acids, hemimellitic acid, trimellitic acid, trimesic
acid.
The atomization can be carried out using all suitable devices; atomization
by means of an ultrasonic nozzle or an aerosol generator is particularly
preferred.
The size of the microcapsules produced according to the invention is
between 50 nm and 500 Vim, in particular between 100 nm and 150 Vim.
The following examples serve to illustrate the invention
Example 1:
Preparation of the solution 1:
9 mg of sodium alginate from Sigma (catalog No.: A-7128) together with
6 mg of BSA-FITC (from Sigma, catalog No.: A-9771 ) are dissolved in 3 ml
of 0.9% NaCI solution.
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Preparation of the solution 2:
1500 ml of 1.0 M hydrochloric acid are heated to 90°C in a 4 I two-
necked
flask, equipped with a condenser. 60 g of chitosan (obtainable under the
name chitosan from Fluka, catalog No.: 22743) are then slowly added with
5 stirring. After addition has taken place, the reaction mixture is stirred at
90°C for 4 hours and then filtered through a G2 frit. The filtrate
obtained is
allowed to stand overnight in a refrigerator at 2-8°C. The precipitate
obtained in this way is isolated by centrifugation (Lobofuge GL from
Heraeus; at 4500 rpm, 25 min). The residue is dissolved in water and
freeze-dried with the aid of a freeze-drying device (LDC-1 m from Christ).
600 mg of the chitosan prepared in this way is dissolved in 30m1 of water
together with 900 mg of CaCl2 (from Riedel deHaen, catalog No.: 12018).
Production of the microcapsules:
3 ml of the solution 1 are atomized at a working frequency of 58 kHz using
an ultrasonic atomizer US2 from Lechler GmbH & Co. KG. With the aid of
carrier air, the spray jet obtained is stabilized to give a spray cone of
about
30° in order to prevent it from being possible for the spray jet to be
influenced by the surrounding atmosphere. In this way, it can effectively be
guaranteed that the drops can bridge relatively large distances. The size of
the liquid droplets obtained in this way is 30 Vim.
This spray cone is directed, at a distance of the nozzle of 3 cm, onto a
glass plate with the dimensions of 5 cm x 10 cm, which is inclined at
30° to
the horizontal. The solution 2 at a rate of 30 Ilh is added above this glass
plate with the aid of a peristaltic pump (from Cole-Parmer Instrument Co.,
model: Masterflex L/STM, tubing LISTM16, speed stage 7). The solution 2
is continuously recirculated in the course of this.
The liquid film comprising the microcapsules is collected in a beaker. After
completion of the atomization process, the microcapsules are separated
from the solution 2 by decantation, washed with 0.9% strength NaCI
solution and stored in this solution. The most widespread microparticle size
is 90 Vim.
Determination of the release of active compound:
For the determination of the release properties of the capsules produced,
BSA-FITC from Sigma (catalog No.: A-9771 ) is used as a model protein.
Further materials are: sodium alginate from Sigma (A-7128), chitosan from
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Fluka (22743), CaCl2 from Riedel de Haen (12018), NaCI from Merck
(6404).
The release measurements are carried out in PBS buffer (Sigma, P4417),
additionally using 0.005% timerosol (from Fluka, catalog No.: 71230).
After production, the PEC capsules are transferred to 10 ml of PBS buffer
solution, in 15 ml rolled-rim vials, and the microcapsules are incubated at
37°C.
The BSA-FITC concentration is measured by means of a UV/VIS
spectrophotometer from Beckmann (DU 70). First, the proportion of
included BSA-FITC is determined by determining the BSA-FITC
concentration in the combined supernatants. The concentration is
determined by measurement of the absorption at 494 nm using a
calibration curve. Falsification of the measurement by the intrinsic
coloration of the chitosan is circumvented by subtracting the absorption of
the chitosan. From the amount of BSA-FITC employed, it can be calculated
how much.
The release measurement is carried out by removing 3 ml from the
incubation solution and determining the BSA-FITC concentration in this
supernatant. After completion of the measurement, the sample solution is
combined again with the release sample. The microcapsules obtained in
this way showed that only 44% of the encapsulated active compound was
released after 30 days.
Example 2 (high-molecular weight chitosan):
The procedure is carried out analogously to Example 1, but the solution 2
is prepared as follows: 90 mg of high-molecular weight chitosan (from
Fluka, catalog No.: 22743) is dissolved in 30 ml of water together with 900
mg of CaCl2 (from Riedel deHaen, catalog No.: 12018) and approximately
100 ~I of acetic acid (Riedel deHaen). The most widespread size of the
microcapsules obtained in this way is 90 Vim; the release of active
compound after 30 days is only 38% of the active compound encapsulated.
Example 3 (crosslinking using glyoxal):
The procedure is carried out analogously to Example 1. After the
production of the microparticles, the particles are crosslinked using glyoxal.
For this, the microparticles are introduced into 10 ml of a 2% strength by
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weight solution for 30 minutes and allowed to stand. They are then washed
with 0.9% NaCI solution.
The most widespread size of the microcapsules contained in this way is
90 Vim.
Example 4 (aftertreatment with pentosan polysulfate)
The procedure is carried out analogously to Example 1. After the
production of the microparticles, the particles are treated with a polyanion
solution. The particles are introduced into 20 ml of a 2% by weight
pentosan polysulfate solution (pentosan polysulfate from Sigma, catalog
No.: P8275) and allowed to stand for 30 minutes. They are then washed
with 0.9% NaCI solution.
The most widespread size of the microcapsules obtained in this way is
90 Vim; the release of active compound after 30 days is only 20% of the
encapsulated active compound.
Example 5 (nanoparticles):
The procedure is carried out analogously to Example 1, but the solution 1
is atomized by a "Pari Inhalierboy" aerosol generator from Pari GmbH. The
size of the liquid droplets obtained in this way is less than 5 Vim. The most
widespread microparticle size of the microcapsules produced is 300 nm.
Example 6 (comparative examplej:
The procedure is carried out analogously to Example 1, but 30 ml of the
solution 2 are introduced into a 250 ml beaker. 3 ml of the solution 1 are
atomized using a US2 ultrasonic atomizer from Lechler GmbH & Co. KG
having a working frequency of 58 kHz. With the aid of carrier air, the spray
jet obtained is stabilized to a spray cone of about 30° and directed
into the
beaker. In this case, caking was observed on the surface of solution 2,
which is to be attributed to uncontrolled crosslinking between alginate and
chitosan. No microcapsules were obtained.
Description of the figures
The following figures show a determination of the particle size distribution
of the microcapsules, produced according to Example 1 (ultrasonic
generator) - Figure 1 - and Example 5 (aerosol generator) - Figure 2 -. In
the case of Example 1, the particle size was determined by Frauenhofer
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diffraction (Cilas granulometer, Cilas 920); in the case of Example 5 by
dynamic light scattering (Malvern Instruments, Mastersizer Microplus).
