Note: Descriptions are shown in the official language in which they were submitted.
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GLASS MICROBEADS HAVING BACTERIOSTATIC PROPERTIES AND
PROCESS FOR MANUFACTU~ING SUCH MICROBEADS
The present invention relates to glass
microbeads having bacteriostatic properties, to a
process for manufacturing them and to their
application in hospital fluidized beds intended for
the treatment of burnt patients.
The bacteriostatic and bactericidal
properties of a number of synthetic or natural
products have been studied for a long time, and some
of these products form part of the traditional
pharmacopoeia, such as antibiotics or disinfectants.
For some years, special beds for the
treatment of individual suffering from severe burns
have been used in hospitals. These beds comprise
fluidized bedding that is to say a mattress or
cushions composed of particles brought to the
fluidized state in a gas such as air and held in
place under a gas-permeable cloth. Sometimes, the
bed simply consists of a steel tank containing the
fine solid particles. The base comprises a blower
which sucks air from the room through a filter. This
air is compressed and heated to a temperature of the
order of 31 to 38C. The air enters a layer of fine
particles, for exemple approximately 25 cm thick, and
brings them to the fluidized state. The particles
are trapped under a gas-permeable cloth made, for
example, of polyester. Any fluids which drain from
the patient can pass through such cloth to form
agglomerates with some particles and such agglomer-
ates are periodically removed by means of a sieve
placed at the bottom of the tank.
It is obvious that bedding of this kind
should not contain or promote the presence of
microbial or bacterial strains. Thus according to
French Patent Application No. 2,523,841 published on
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September 30, 1983, it has been proposed to add to
particles such as glass microbeads forming the
fluidized medium, particles of another material
having bactericidal properties. The particles
proposed are particles of metal such as calcium or
magnesium or aluminium or bismuth, or silicon
particles. Apart from difficulties which may arise
from the choice of the size of such particles in
order to render them capable of fluidization without
segregation from the glass microbeads, it is obvious
that a proposal of this kind possesses a definite
risk in use. There is a risk of ignition of the
particles in the presence of moisture or of a hot
spot, or even at room temperature in the case of
calcium. It is therefore desirable to find other
means which are simpler and safer for endowing the
said
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bedding with bactericidal or bacteriostatic properties.
According to the present invention, glass microbeads are
characterized in that the beads are coated with proteins which are bound
covalently to the glass and en<low the beads v~tith bacteriostatic properties.
s The present invention thus provides an answer to the problem of
endowing the said bedding with bactericidal or bacteriostatic properties, since it
enables the use of a single type of particles to be introduced into hospital
auidized beds, namely glass rnicrobeads which in themselves possess bacteriostatic
properties. Furthermore, such particles can be recycled and regenerated.
o The microbeads according to the invention, to which proteins which
provide them with a bacteriostatic power are bound covalently, retain their
properties with the passage of time; these properties are maintained for severalmonths before use if the beads are stored under good conditions, in dry, sealed
packaging, and they retain their bacteriostatic power for a period compatible with
their use in fluidized beds, that is to say for several days or even several weeks
exposure to the air. This bacteriostatic power is maintained when the beads are in
a damp atmosphere or when they are subjected to a moderate temperature (for
example below 60 or 70C). The bacteriostatic power is preserved even when the
beads are subjected among themselves to abrasion during their handling and use.
This is thought to be due to the covalent bond existing between the proteins andthe glass. Altogether unexpectedly, it is found that, despite the fact that the said
proteins are bound to the glass via a covalent bond, the proteins retain their
bacteriostatic properties and are capable of imparting such properties to the
microbeads which support thenL
As already stated above, the rnicrobeads possess bacteriostatic
properties and, in many cases, they are also bactericidal, depending on the nature
of the proteins and their concentration. By way of example, it is very easy to
render them capable of killing or lirniting the growth and multiplication of
bacteria such as Escherichia coli, Salmonellae, Pseu~lomonas, Legionellae and
Staphylococcus aureus.
The microbeads according to the invention may be used in direct
contact with the skin, for example in dressings, and in this case a special
biodegradable glass will preferably be selected. It is also possible to envisagebinding to the microbeads proteins which make them active, from the bactericidal3s standpoint, in the digestive tract of human beings and animals. It is also possible
to use them for creating a sterile medium out of contact with the body, as is the
case for fluidized bedding. Given the importance of this latter use, the present
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description refers especially to the it, but it is to be understood that the present
invention is not exclusively limited to that use.
After the rnicrobeads according to the invention have been In use for
a certain period of time, it may be necessary or desirable to regenerate them for
s reuse. For example it will generally be necessary to change the bea~ls of fluidized
bedding for the treatment of burn patients when a new patient is transferred to the
bed. Used beads can readily be sterilized by a heat treatment. For example they
may be treated at a temperature of about 100C for a sufficient length of time.
Such a treatment also removes the protein coating from the beads, but a fresh
lo coating of protein may readily be covalently bonded to the beads so that they may
be reused.
In the most preferred embodiments of the invention, the said
microbeads have a non-porous surface. The adoption of this feature presents
considerable advantages from the point of view of hygiene. Any body, or other,
15 fluids which come into contact with beads having porous surfaces may readily be
adsorbed. Though this adsorbed material can easily be sterilized as referred to
above, it is extremely difficult to remove from porous beads, so that it remains as a
possible health hazard, even ffla steriL;zation and bonding of bacteriostatic
proteins to the beads. If the bacteriostatic coating of such a porous bead should
20 faiL the adsorbed material may be a fertile microbiological breeding ground. This
is not the case with beads having non-porous surfaces. Very little, if any, of such
material is found to become adsorbed, and what is adsorbed is easily removed
during sterilization so that it is possible to reuse the beads with much less risk of
hazard to the patient. Beads having non-porous surfaces have the further
25 advantage over beads with porous surfaces that they are in general easier and therefore 1ess expensive to produce.
Preferably, the proteins which are bound to the glass microbeads are
enzymes. The choice of enzymes enables the growth or multiplication of bacteria
to be suppressed or prevented according to an altogether selective specific
30 mechanism enabling an entity detrimental to bacteria to be created in situ. The
preferred enzymes for application to ~uidized beds are peroxidases, which catalyse
the oxidation of various ions present in fluids such as plasma or serum, creating
bactericidal entities.
Proteins such as transferrin or myeloperoxidase may be bound to the
35 beads, but it is preferable to select proteins such as lactoferrin or lactoperoxidase.
Lactoferrin takes up iron ions and thereby creates a medium unsuitable for
bacterial growth. Lactoperoxidase, with an oxidizing agent such as hydrogen
132~2Q9
peroxide (provided metabolically or by the action of glucose oxidase on glucose)and SCN ions, forms a medium containing OSCN ions which destroy bacteria.
Since the bacteriostatic action of proteins is very efficient, it is not
necessary cornpletely to coat the surfaces of the beads with proteins. Preferably,
s the proteins are present in the proportion of less than 0.1~ by weight with respect
to the weight of the glass. A quantity as sma11 as 0.05% by weight is effective, and
it is often sufficient to use a quantity of 0.025'o.
As stated above, a great advantage of the microbeads according to the
invention resides in the fact that the latter retain their bacteriostatic or
o bactericidal properties despite the mechanical and thermal stresses to which they
may be subjected. This is probably linked to the existence of a covalent bond
between the proteins and the glass. To facilitate covalent attachment of the
proteins to the glass, it is preferable to use a coupling agent capable of creating, at
the surface of the glass, preferential attachment sites for the reactive groups of the
s proteins: These reactive groups consist of amino acids. As a coupling agent, it is
possible to use an organic titanate, but it is preferable to select a silane type
coupling agent, since the range of silanes offers a wide choice of substances
capable of reacting directly with amino acids.
By way of a variant, it may be preferable to bind the proteins by
20 means of a silane and a second coupling agent. In this case, the silane creates an
aminated glass surface which is linked to the proteins via a coupling of the
"Michael" type, with glutaraldehyde, or of the amide type, for example with
succinic anhydride, or of the æo type, for example with nitrobenzoyl chloride. It
is also possible to create a thioester bond in the coupling chai4 which has the
25 advantage that it can be readily cleaved if it is desired to separate the beads from
the proteins for the purpose of recycling. Coupling with glutaraldehyde is
advantageous, since it permits the rapid binding of a wide range of proteins or
different enzymes to the glass.
Preferably, the microbeads according to the invention are
30 hydrophobic. This property enables them to keep their integrity in the presence
of moisture or of various physiological ~uids, and makes it possible, above all, to
prevent them from agglomerating in the presence of these fluids. The
hydrophobic rnicrobeads according to the invention preferably owe their
hydrophobic nature to the presence of a silicone coating. A silicone which
3s polymerizes at the free surface of the glass without binding thereto via covalent
bonds is preferably selected. For example, an amino-group-containing
polydimethyl- siloxane copolymer such as the product Dow Corning MDX4-4159,
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which polymerizes at room temperature or moderate temperature, may be used.
A silicone of this kind is compa~ble with the presence of proteins and,
surpnsingly, does not modify or only neglig~bly modifies the bacteriostatic activity
of the microbeads with respect to identical rmicrobeads coated only with proteins.
s The glass microbeads according to the invention are solid glass
microbeads or hollow rnicrobeads. For use in hospital fluidized beds, it is
preferable that these microbeads have a relative density greater than 1, in order to
facilitate the supporting of the patient, though hollow microbeads have the
advantage of requiring a smaDer quantity of fluidization air, resulting in less
lo drying-out of the fluidization atmosphere.
Microbeads are preferably selected whose particle size distribution is
narrow, which facilitates their fluidization without segregation. For example,
glass microbeads whose diameter lies between 65 and 110 micrometres are chosen.
It is possible to use microbeads having an average diameter which lies between 80
IS and 100 micrometres, and preferably between 85 and 90 rnicrometres. Suchbeads do not require too much energy for their ~uidization, and are not capable of
passing through the meshes of the cloths normally used for hospital fluidized beds.
For this particular application, solid glass microbeads or hollow microbeads maybe selected.
The present invention also covers microbeads having bacteriostatic
properties suspended in a fluidization gas, and encompasses a bed for the
treatment of burn patients comprising a fluidization system containing rnicrobeads
as defined above.
The present invention also relates to a process for endowing glass
rnicrobeads with bacteriostatic properties, characterized in that a coating of
proteins that are bound covalently to the glass is attached to these rnicrobeads. A
process of this kind has the advantage of obtaining a finely divided particulatematerial that is readily capable of fluidization and possesses and retains effective
bacteriostatic properties. The process incorporates a stage during which the
microbeads are brought into contact with proteins, for example in suspension or
powder form
Advantageously, the stage of bringing the microbeads into contact
with the proteins is preceded by a stage of binding a coupling agent to the surface
of the glass. The coupling agent is preferably a silane which deposits readily on
the surface of the glass from a solution, suspension or powder, at room
temperature. It is also possible to graft, for example, at the surface of the glass,
amino or oxirane groups capable of reacting with the amino acids of the proteins.
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In some cases, it may be useful to treat the glass ~th a second
coupling agent. This is the case, for example5 if the silanization of the glass
creates amino groups at the surface of the latter. In this case, the protein is bound
via a coupling of the "Michael" type with glutaraldehyde, or of the amide type or
s the azo type. In order to avoid denaturation of the proteins, it is recornmended to
perform the binding of the proteins to the glass by bringing the latter into contact
with a medium containing the said proteins in which the pH does not exceed 7. A
medium containing the proteins in which the pH is between 4 and 5 is preferably
selected.
o Preferably, after the said proteins have been bound to the glass, themicrobeads are brought into contact with a medium containing a silicone, in order
to deposit a silicone coating on the beads. The beads thus treated are
hydrophobic and have no tendency to agglomerate. The medium containing the
silicone preferably has a pH of between 4 and 5. The deposition of silicone is
s carried out at room temperature or moderate temperature. In general, in ordernot to denature the proteins, it is recommended to deposit and, where
appropriate, to polymerize the silicone at a temperature not exceeding 60 to 70C.
When the microbeads have been used for a certain length of time, for
example during a change of patients in a fluidized bed for the treatment of burn20 cases, it may prove necessary to regenerate the microbeads. In order to do this, it
is desirable first to sterilize the used beads. Such beads may easily be sterilized by
a heat treatment, for example by treating them at a ternperature of the order of100C for sevaal hours. This treatment removes the covalently bound proteins
but retains the silicone at the surface of the glass. Accordingly, the invention also
2s encompasses a process for restoring the bacteriostatic properties of glass
microbeads, characterized in that the microbeads which have been sterilized by aheat treatment are then treated by a process of covalent binding of proteins as
described above.
The present invention will be explained in greater detail with
30 reference to the examples which follow.
Example 1
Solid alkali-lime glass microbeads are selected by sieving so as to
remove the beads smaller than 65 micrometres and larger than 106 micrometres.
The average diarneter (by weight) of the beads selected is 8S micrometres.
The microbeads are - treated with (gamma-
glycidoxypropyl)trimethoxysilane (A 187 of Union Carbide) in a1coholic solution
at room temperature, in the proportion of 0.1 cc of silane per kilogramme of
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beads, which corresponds to approximately 100 mg of silane per kilogramme of
beads.
Lactoferrin is then bound covalently to the beads. The lactoferrin is
bound via its amino acids to the epoxy groups of the silane. This operation takes
s place at room temperature, by bringing the silane-treated beads into contact with
a 10% strength aqueous solution of bovine lactoferrin marketed by Oléofina, at apH between 4 and 5. There is thus approximately 0.01% by weight of active
product with respect to the weight of the glass.
The microbeads are then gently heated, care being taken not to
o exceed 60~C, and they are brought into contact with a silicone fluid. The silicone
selected is a Dow Corning amino group containing polydimethylsiloxane
copolymer MDX4-4159, which is used in the proportion of 0.2 cc per kilograrnme
of glass, which corresponds to approximately 200 mg of silane per kilogramme of
beads.
s The bacteriostatic power of these microbeads is identical to that oflactoferrin in solution, that is to say not imrnobilized. It has been found, forexample, that these microbeads inhibit the growth of an E coli strain.
These microbeads are hydrophobic, and may be stored, manipulated
and used without a risk of agglomeration.
Example 2
Alkali-lime glass microbeads similar to those of Example 1 are treated
using Union Carbide (gamma-aminopropyl) trimethoxysilane A1100, in alcoholic
solution, in the proportion of 0.1 cc of silane per kilogramme of glass, which
corresponds to approximately 100 mg of silane per kilogramme of glass. The
2s surface of the beads is then activated by reacting the silane with glutaraldehyde, at
a pH below 6.5, in stoichiometric proportions.
Lactoperoxidase is then bound to the beads by bringing the latter into
contact with an aqueous solution of lactoperoxidase, marketed by OléoSna.
0.02% by weight of lactoperoxidase with respect to the glass is thereby bound
,' 30 covalently. A silicone identical to that of Example 1 is then deposited on the
surface of the beads.
It was found that the lactoperoxidase retained its enzymatic activity
despite its covalent binding to the glass. This enzymatic activity was assayed by
the o-phenylene-diarnine method, and a value of 350 U/rng of lacto peroxidase
3s bound to the glass was observed, while the same method applied to an equivalent
quantity of lactoperoxidase in solution had an activity of approximately 400 U/mg
of enzyme (U/mg = unit of specific activity of the protein per rng; one unit of
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activity is the quantity of enzyme which produces, in one minute, an increase inabsorbance at wavelength 490nm of 0.1, at 37C and at pH 5, and with o-
phenylene-diarnine and H2O2 as substrate).
The bacteriostatic power of the microbeads was also examined. This
s bacteriostatic power was assessed with respect to an E. coli strain sensitive to the
action of OSCN- ions. The change with respect to time in the optical density of
such a culture at wavelength 660 nm was examined. An increase in the optical
density is evidence of the proliferation of bacteria In the absence of
lactoperoxidase, a control sample shows an increase in optical density
o corresponding to a multiplication of the initial value by a factor of the order of 100
after 6 hours at 37C In contrast, in the presence of 8 8 per litre of beads treated
according to the present example (which corresponds to 500 U of lactoperoxidase
per litre of culture medium), the optical density is unchanged after 6 hours, which
demonstrates blocking of the bacterial growth.
s By way of comparison, microbeads treated as described above but not
bearing silicone were brought into contact with the same E. coli strain. It is found
that, for an identical quantity of lactoperox~dase bound to the beads, the activity of
the beads coated with lactoperoxidase and silicone is virtually identical to that of
the unsiliconized beads. There is hence, surprisingly, no interference between th
activity of the immobilized enzyme and the presence of the silicone.
Similar results are found with other bacteria such as Pseudomonas
and Staphylococcus aureus.
ample 3
In the first place, (gamrna glycidoxypropyl)trimethoxysilane is bound
2s as described in Example 1 to glass rnicrobeads identical to those of Example 1, and
0.05% by weight of lactoperoxidase is then bound directly to the microbeads. Thetreatment is cornpleted by depositing silicone identical to that mentioned in
Examples 1 and 2.
The test of culturing bacteria of Example 2 was repeated, and it was
found that 7 g of beads per litre of culture medium sufficed for blocking growth of
the bacteria.
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