Note: Descriptions are shown in the official language in which they were submitted.
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Process for the roduction of collaaen barticles and their
Use as Carriers for ~rctive Substances
DR$CRIPTIUN
The invention concerns a process for the production of
collagen particles whose size lies in the micro and manome-
ter range, and their use as carriers for active substances
in medicine, the food processing industry and cosmetics.
The inclusion of a great many active substances and other
components such as dyestuffs, catalysts, enzymes, radioac-
tive material, flavours, volatile compounds and the like in
just as many kinds of casing materials in capsules of a
size lying in the micro and manometer range is well-known.
In the pharmaceutical sphere distilled oils in gelatine
cases, for example, are used as aroma carriers, which only
release the aroma when the case has dissolved.
The purpose of the encapsulation is - in addition to effec-
ting the release of the contents after the case has dis-
solved - for example the protection of active substances
against oxygen, moisture or other. chemical agents; the
transformation of a fluid into a "dry" powder; the produc-
tion of depot forms of active substances by means of the
appropriate treatment or the choice of relevant casing
material; or the production of complexes of active substan-
ces and adjuvants by loading the surface of the carrier
particles with active substances.
Particle sizes in the manometer range offer, in addition to
this, the possibility - as a result of the extremely small
dimensions of the capsules - of dissolving the latter
colloidally in water together with the enclosed or absorbed
active material. It is thus possible to administer such
solutions intravenously as well.
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Collagen is the main component of the connective tissue in
vertebrates and is almost ubiquitous.
Particulate carriers of drugs made of collagen possess the
advantage, compared with other carrier systems, that. owing
to the biocompatibility and the complete biodegradation and
metabolic conversion of the particles, toxicological
problems are not to be expected when they are applied
locally and systemically.
The use of collagen as a capsule encasing material offers
the additional advantage that, in the case of substances
possessing a high protein affinity, which bind very well
with protein particles, a high loading of the carrier
system is possible.
The capacity of the carrier system for adsorbent loading
is, in these cases, a great deal higher than that of, fox
example, carrier particles on an acrylate basis, taking the
same particle surface area into account.
Processes for the production of collagen microparticles are
described, for example, in gS Patent Specification 4 565
580 and in TJS Patent Specification 4 107 288. The expres-
sion ''microparticle'° is used in the sense that hereby
particle sizes in the micrometer range and smaller are
described.
A common feature of the processes described there is that ,
the microparticles are produced according to the principle
of coacervation.
In this process, desolvating agents, such as, for example,
alcohols or electrolytes, are added to a molecular disper-
sed solution of collagens. The solubility of the protein
is reduced by means of the admixture, which results in a
cloudiness of the solution, indicating the formation of
light-scattering protein aggregates.
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Tf one separates the coacervate and dries it, in the end
one obtains microcapsules, which can be hardened by a
subsequent treatment. The particle diameters are in the
range of 10 - 1000 ~tm (OS Patent Specification 4 565 580)
and 10 - 1000 nm (US Patent Specification 4 107 288),
respectively.
In US Patent Specification 4 837 285 a process is described
in which an aqueous collagen dispersion is forced through a
vibrating, hollow tube whose diameter is defined. Droplets
form at the end of the tube, which drip into a cooling bath
comprised of liquid nitrogen. After the nitrogen has
evaporated, the frozen droplets are dry frozen and then
cross-linked in an aqueous dispersion, so that they are
aftercured. The particle diameter is between 100 and 400
N.m .
The above-mentioned collagen microparticle size distri-
butions according to processes of the prier art already
show the serious disadvantages. Por one thing, the par-
ticle size distribution is spread over a very wide range.
Homogeneous particle fractions with a very narrow particle
distribution cannot be obtained according to this process,
as the agglomeration of the collagen molecules and the
particle agglomeration happen at random and can scarcely be
controlled.
Defined collagen particle fractions with a diameter in the
low micrometer and in the manometer range cannot be ob-
tained, according to this process, in particular. so that
the microparticles produced according to this process are
unsuitable for preparations for intravenous injection.
The present invention takes as its basis the task of fin-
ding a process for the production of homogeneous callagen
particles in the micrometer and manometer range, with a
narrow particle size distribution to be controlled by the
process.
The task is solved surprisingly by a process in which a
solution or dispersion of collagen in water is distributed
finely in discrete droplets in a waterimmiscible organic
phase, whereby emulsifying agents are added, a water-in-oil
emulsion thereby being formed, t:he collagen being subse-
quently cross-linked in the interface of the droplets,
using a cross-linking agent, and the collagen particles
produced in this manner being purified and isolated by
separating the organic phase and washing.
To produce the particles, an emulsion is prepared in a
manner known to a person skilled in the art, from an a-
queous collagen dispersion, which can contain native col-
lagen or collagen which has been denatured by means of
chemical agents or physical influences from sources of
various origins known to the man of the art, from an emul-
sifying agent and from an organic phase. The determination
of the emulsion type can take place by means of conduc-
timetry.
Emulsifying agents which can be employed to advantage in
the processes according to the inventions are polyethylene-
glycol fatty acid esters, such as, for example. polyethy-
leneglycol-400-stearate: polyethyleneglycol fatty alcohol
ethers, such as, for example, polythyleneglycol-200-lauryl
ether; polyethyleneglycol sorbitane fatty acid esters, such
as, for example, polyethyleneglycol-~~0)-sorbitane mono-
oleate; partial fatty acids of polyhydric alcohols, such
as, for example, glycerol monostearate or sorbitane tri-
oleate; or partial fatty acid esters of sugars, such as,
for example, saccharose monolauryl acid ester.
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The waterimmiscible organic phase can contain natural,
halfsynthetical or synthetical liquid fats, oils or waxes,
such as, for example, olive oil, castor oil, cotton seed
oil, soya bean oil, hydrated peanut oil, triglyceride
mixtures (Miglyol~, Softisan~), silicone oil, oleic acid
oleyl ester, isopropyl myristate or ethyl oleate.
The production of the emulsion is the decisive step
influencing the size and homogeneity of the microparticles
which ultimately result. The size of the dispersed water
droplets in the organic phase is determined and controlled
by both the kind of emulsifying agent or emulsifying
complex and the kind and intensity of the emulsifying
process.
The collagen molecules are then polymerized by adding a
cross-linking agent in the interface of the droplets.
Mono- or bifunctional aldehydes such as, for example,
formaldehyde, glutaric dialdehyde or dialdehyde starch; or
bifunctional isocyanates, such as, for example, hexa-
methylene diisocyanate, can be used as cross-linking
agents.
The organic phase is subsequently separated and the par-
ticles purified by washing, i.e. the remainders of the
organic phase, the emulsifying agent and the cross-linking
agent are got rid of.
It may be necessary to redisperse the collagen micropar-
ticles in the ultrasonic bath, to break up the aggregates.
The determination of the particle size and the size distri-
bution take place after freeze drying and redispersion by
means of photon correlation spectrometry. At the same
time, a laser beam is sent at 20 impulses per second
through a vessel filled with a dilute, colloidal solution
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of collagen particles. The laser light, being scattered by
the particles, is registered via a photoelectric cell at an
angle of 90° to the irradiating laser.
The scattering is dependent on the size of the particles;
by conversion of the electric signal obtained, it is pos-
sible to infer the particle size by means of a mathematical
equation.
Because of the Brownian movement, at every impuls another
"particle population'° is hit. In this manner the infor-
mation gained from the large number of impulses (r 2500)
provides an exact picture of the average size and size
distribution of the particles.
Active substances can be added to the emulsion prior to the
cross-linking of the particles.
The active substances axe enclosed in the particles during
production of the particles by interface polymerisation, or
are adsorbed at the surface of the particles after particle
formation.
If the loading with active substances is to take place
after the production of the particles, the collagen par-
ticles are, after their production, separation and purifi-
cation, subjected to any desired drying process known to
the man skilled in the art, preferably using the freeze
drying process.
At a high temperature reduction rate the particles are
shock-freezed in a freeze-dryer to temperatures <_ -50°C, so
as to form ice crystalls which are as small as possible,
and are subsequently freeze-dried under vacuum.
The drying process increases the stability of the particles
during storage until further processing.
For subsequent loading of the dried particles with active
substance, the particles are first redispersed in water;
then the active substance. an active substance solution or
active substance dispersion is added to the dispersion.
The particles, loaded with active substance through ad-
sorption, are subsequently separated and purified.
According to the process of the present invention, it is
possibl~, by means of emulsion polymerisation, to produce
collagen particles with very small diametres which can be
used in preparations for intravenous injections or for
application on the eye; the comparatively simple process
allows accurate process control and thus improved control
of the production of homogenous amounts of particles with
the respective, desired narrow size distribution and at
high yields.
The invention is illustrated by means of the following
examples:
Example 1:
Production of collagen microparticles from a water-in-oil
emulsion:
By means of a pestle, 15 g of sorbitane monolaureate are
homogenously incorporated into 100 g of cotton seed oil,
which has been put into a mortar. After 10 minutes, 25 g
of a 1 p. c. dispersion of native calf skin collagen is
added in five portions and is carefully incorporated. The
emulsion is homogenised for 3 x 10 minutes in the ultraso-
nic bath, while being cooled.
In a further process step the emulsion is treated with a
high-speed homoginizing mixer for 3 x 15 seconds, with
intermediate cooling. The emulsion is stirred at 400 rpm
onto a magnetic stirrer; to effect the cross-linking of the
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particles, 5.0 m1 of glutaric aldehyde solution (25°'°) are
added. After 10 minutes, the reaction is stopped by adding
7.0 ml of Na-disulfite solution (25°~), and the mixture is
thereafter worked up.
An excess of ether is added three times to the preparation
and the latter is centrifuged for 10 minutes each time, at
2500 rpm; this process is repeated twice with an excess of
water. The particles are either freeze-dried or are im-
mediately processed further.
In the present example, collagen particles with an average
diametre of 2.15 ~tm were obtained; 90 °~ of the particles
were smaller than 9,2 Nm.
The collagen particle yield was ca. 95%, relative to the
amount of pure collagen used.
Example 2:
The experiment was carried out as described in example 1,
whereby instead of a 1 p, c. dispersion of native calf skin
collagen, a 1 p. c. dispersion of denatured calf skin
collagen was used.
The average particle diametre obtained was 320 nm.
Yield: 90 %.
Example 3:
Production of collagen microcapsules from a water-in-oil
emulsion
g of polyoxyethylene-(20)-sorbitane monooleate are
dispersed in 25 m1 of a 0.75 p. c. dispersion of native
calf skin collagen. After 10 minutes 100 g of cotton seed
oil are added in five portions and stirred. Each time. the
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emulsion is subsecguently worked for a period of 5 minutes
with the high-speed homogenizing mixer and the slit homoge-
nizer.
1.0 g of glutaric aldehyde solution (25%) is then added and
the dispersion is stirred for 10 minutes. To recover the
excess of glutaric aldehyde, 1.4 ml of Na-disulfite
solution (25%) are added. The removal of the organic phase
is carried out as described in example 1. Yield: 92 °%.
The particles obtained exhibited an average diametre of
1.23 Vim. 90 % of the particles were smaller than 2.2 ~tm.
Exam~l~ 4:
Loading of the collagen particles with ethacridine lactate
50 mg of particles are dispersed in 50 ml of water, in the
ultrasonic bath. 25 mg of ethacridine lactate are added to
the particle dispersion and stirred for 24 hours at 200
rpm.
To determine the load, the suspension is ultrafiltrated;
the content of medicinal agent in the supernatant is deter-
mined, and subsequently the particle load is calculated.
The particle load is 12 mg on 50 mg of collagen particles.
The absolute load is thus 24 °~-wt, relative to the medi-
cinal agent used.
Example 5:
Loading of the collagen particles with chlorophyll
250 mg of collagen particles are dispersed in 22,5 ml of
physiological NaCl solution. The dispersion is exposed to
sonic waves for 10 minutes. Thereafter, 2.5 ml of satu-
rated ethanolic chlorophyll solution are added and stirred
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for about 2~4 hours at 300 rpm. The particles are purified
in a GPC column, which is filled with Sephadex~ G-50
(arose-linked polysaccharide).
A second band does not appear, which indicates that the
loading was carried out quantitatively. The particles
obtained are freeze-dried.
