JPH0193444A - Glass microbeads having bacteriostatic properties and method for producing such microbeads - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to glass microbeads with bacteriostatic properties, a method for their production and their application in hospital fluidized beds for the purpose of treating burn patients.

The bactericidal and bactericidal properties of a large number of synthetic or natural products have been studied for a long time, and some of these products form part of traditional pharmacy, such as antibiotics or fungicides.

For several years, special beds have been used in hospitals to treat individuals with severe burns. These beds include a fluidized bed of particles held in place under a gas-permeable fabric and kept fluidized in a gas such as air. Sometimes the bed simply consists of a steel tank containing microscopic solid particles. The base has a blower that sucks air from the chamber through a filter. This air is compressed and heated to a temperature on the order of 31-38°C. Air is composed of minute particles, for example, about 25 cm thick.
enters the layers of rn and brings them to a fluidized state. The particles are trapped under a gas permeable fabric made of polyester, for example. Fluid expelled from the patient passes through such fabric and forms agglomerates with particles, which are periodically removed by a sieve placed at the bottom of the tank.

It is clear that beds of this type must not promote or contain the presence of microorganisms or bacterial strains. According to French Patent Application (FR) No. 2,523,841, it was therefore proposed to add particles of another material having bactericidal properties to the particles, such as glass microbeads, forming the fluidized medium. The proposed particles are particles of metals, such as calcium or magnesium or aluminum or bismuth or silicon particles. Apart from the difficulties that may arise from the choice of the size of such particles, in order to be able to fluidize them without segregation from the glass microbeads, proposals of this kind have It is clear that there is a risk of There is a risk of particle ignition in the presence of moisture or hot spots, or even at room temperature in the case of calcium. It would therefore be desirable to find other simple and safe means of providing said bed formation with bactericidal or bactericidal properties.

According to the invention, the glass microbeads are characterized in that they are coated with a protein that covalently binds to the glass and imparts bacteriostatic properties to the beads.

The present invention provides an answer to the problem of providing said bed formation with bactericidal or bacteriostatic properties, since a simple type of beads to be introduced into the hospital fluidized bed, i.e., which is itself bacteriostatic. This is because it allows the use of glass microbeads. Moreover, such particles can be recycled and regenerated.

The microbeads according to the invention, to which proteins providing bacteriostatic power are covalently attached, retain their properties over time, and the beads are dried and stored under good conditions in sealed packages. In other words, if you maintain these properties for several months before use and use them in the form of a fluidized bed,
They maintain their bactericidal activity for days or even weeks upon exposure to air. This bacteriostatic power is maintained when the beads are in a humid atmosphere or when they are exposed to moderate temperatures (eg less than 60°C or 70°C). The beads retain their bacteriostatic power even when they undergo abrasion among themselves during their handling and use. This is thought to be due to the covalent bond that exists between the protein and glass. Quite surprisingly, despite the fact that the proteins are bound to the glass via covalent bonds, the proteins retain their bacteriostatic properties and transfer such properties to the microbeads supporting them. It is found that it is possible to give.

As mentioned above, microbeads have bacteriostatic properties, and in many cases they are also bactericidal depending on the type of proteins and their concentration. For example, Niskelizia fistula (Es
oheriohia coli), Salmonella (Sa
lmonellae), Pseudomonas (Psaud
omonag), Legionella
e) and Histahirokotsucus 6 aureus (Staph
It is very easy to inhibit the growth and proliferation of bacteria such as Yloooceusaursus) or to be able to kill them.

The microbeads according to the invention can be used in direct contact with the skin, for example wrapped in a bandage, in which case special biodegradable glass is preferably selected. From a sterilization standpoint, microbeads can also be bound to proteins that make them active in the gastrointestinal tract of humans and animals. They can also be used to create sterile media that come into contact with the body, such as for fluidized beds. Because of the importance of this latter use, the description of the invention will be presented with particular reference thereto, although it should be understood that the invention is not limited exclusively to that use.

After using the microbeads according to the invention for a period of time, it may be necessary or desirable to regenerate them for reuse. For example, when transferring a new patient to a bed, it is generally necessary to change the beads in a fluidized bed for the treatment of burn patients. The beads used can be easily sterilized by heat treatment. For example, they can be treated at a temperature of about 100° C. for a sufficient length of time. Such treatment also removes the protein coating from the beads, but new protein coatings can easily be covalently attached to the beads, so they can be reused.

In the most preferred embodiment of the invention, said microbeads have a non-porous surface. The adoption of this feature offers significant advantages from a hygienic point of view. Body fluids that come into contact with beads having porous surfaces can be easily adsorbed. Although this adsorbed material can be easily sterilized as mentioned above, it is very difficult to remove it from the porous beads, so even after sterilization it is difficult to bind bacteriostatic proteins to the beads. Even after it has been removed, it can still pose a health risk.

When the bacteriostatic coating of such porous beads fails, the adsorbed material can become a medium for microbiological growth. This is not the case for beads with non-porous surfaces. It is found that very little, if any, such material becomes adsorbed, and that what is adsorbed is easily removed during sterilization and therefore poses very little risk to the patient, and the beads can be reused. Beads with non-porous surfaces are generally easier and therefore less expensive to make, and have advantages over beads with porous surfaces.

Preferably, the protein bound to the glass microbeads is an enzyme. The selection of enzymes makes it possible to inhibit or inhibit the growth or multiplication of bacteria by a totally selective special mechanism that makes it possible to create in situ something that is actually harmful to the bacteria. A preferred enzyme for use in fluidized beds is peroxidase. It catalyzes the oxidation of various ions present in fluids such as plasma or serum to create bactericidal entities.

Transferrin or myeloperoxidase
Proteins such as lactoferrin or lactoperoxidase can be bound to beads, but proteins such as lactoferrin or lactoperoxidase
Preferably, proteins such as e) are selected. Lactoferrin absorbs iron ions, thereby creating an unsuitable medium for bacterial growth. Lactoperoxidase, with an oxidizing agent such as hydrogen peroxide (provided metabolically or by the action of glucose oxidase on glucose) and 5CN- ions, forms a medium containing 03CN- ions that destroys the bacteria.

Since the bacteriostatic action of proteins is very efficient, it is not necessary to completely coat the surface of the beads with proteins. Preferably,
The protein is present in a proportion of less than 0.1% by weight based on the weight of the glass. Amounts as low as 0.05% by weight are effective, and amounts of 0.02% by weight are often sufficient.

As mentioned above, the advantages of the thick microbeads according to the present invention are that
The fact is that they retain their bacteriostatic or sterile properties despite the mechanical and thermal stresses to which they may be subjected. This is probably related to the presence of covalent bonds between the protein and the glass. To facilitate covalent bonding of proteins to glass, it is preferred to use coupling agents that can create selective bonding sites for reactive groups of proteins at the surface of the glass. These reactive groups consist of amino acids. Although organotitanates can be used as coupling agents, it is preferable to choose silane-based coupling agents, since the range of silanes provides a wide selection of substances that can react directly with amino acids. It is.

As a modified example, proteins can also be bonded by silane and a second coupling agent, which is preferred. In this case the silane is coupled to the protein via a Michael-type coupling with glutaraldehyde, or via an amide-type coupling with e.g. succinic anhydride, or via an azo-type coupling with e.g. evanitrobenzoyl chloride. Make aminated glass. It is also possible to create a thioester bond in the coupling chain.

This has the advantage that it is easily cleaved when it is desired to separate the beads from the protein for recycling. Camping with glutaraldehyde is advantageous because it allows rapid binding of a wide range of proteins or different enzymes to the glass.

Preferably the microbeads according to the invention are hydrophobic. This property makes it possible to maintain their integrity in the presence of moisture or various physiological fluids and, among other things, to prevent them from condensing in the presence of these fluids. Hydrophobic microbeads according to the invention preferably derive their hydrophobicity from the presence of a silicone coating.

Preferably, silicones are selected that polymerize on the free surface of the glass without bonding to the glass via covalent bonds. For example, amino group-containing polydimethylsiloxane copolymers such as Dow Corning product MDX 4-4159, which polymerize at room or moderate temperatures, can be used. This type of silicone is compatible with the presence of proteins and, surprisingly, has only minimal or negligible denaturation of the bacteriostatic activity of the microbeads against the same microbeads coated only with protein. do not have.

The glass microbeads according to the invention are solid glass microbeads or hollow microbeads. For use in fluidized beds in hospitals, these microbeads preferably have a relative density of 1 to 1 to facilitate patient support.
However, hollow microbeads have the advantage of requiring less fluidizing air, making the fluidizing atmosphere less dry.

Preferably, the microbeads are selected with a narrow particle size distribution, which facilitates their fluidization without segregation. For example, glass microbeads with a diameter of 65 to 110 μm are selected. 80-100μm, preferably 85-100μm
Microbeads with an average diameter of between 90 μm can be used. Such beads do not require too much energy for their fluidization and cannot pass through the fabric mesh normally used for hospital fluidized beds. For this particular application, solid glass microbeads or hollow microbeads can be selected.

The present invention also includes microbeads with bacteriostatic properties suspended in a fluidizing gas, and includes a bed for the treatment of burn patients that includes a fluidization device containing microbeads as described above.

The invention also relates to a method for making glass microbeads with bacteriostatic properties, which is characterized by attaching to these microbeads a coating of a protein that is covalently bound to glass. This kind of method can be easily fluidized,
It has the advantage of obtaining a micronized material that has and retains effective bacteriostatic properties. The method incorporates the step of bringing microbeads into contact with a protein, for example in suspension or powder form.

Advantageously, the step of bringing the microbeads into contact with the protein is preceded by a step of attaching a coupling agent to the surface of the glass. Preferably, the coupling agent is a silane that readily adheres to the glass surface from solution, suspension or powder at room temperature. It is also possible to graft, for example, amine groups or oxirane groups on the surface of the glass, which can react with amino acids of proteins.

In such cases, it is also useful to treat the glass with a second coupling agent. This is the case, for example, when silanization of glass creates amino groups on the surface of the glass. In this case, the protein is coupled with glutaraldehyde via a Michael-type coupling or via an amide-deaf or azo-type coupling. In order to avoid denaturation of the proteins, it is recommended to bring the glass into contact with a medium containing said proteins at a pH not exceeding 7, and to carry out binding of the proteins to the glass. Preferably, a medium containing proteins with a pH of 4 to 5 is selected.

Preferably, after binding the protein to the glass, the microbeads are brought into contact with a medium containing silicone in order to deposit a silicone coating on the beads. Beads thus treated are hydrophobic and have no tendency to stick. Preferably, the silicone-containing medium has a pH of 4 to 5.

In general, it is recommended that the silicone be deposited or, if appropriate, polymerized, at temperatures not exceeding 60-70° C. in order not to denature the protein.

It is clear that when the microbeads have been used for a certain period of time, it is necessary to regenerate them, for example during patient changes in a fluidized bed to treat burns. To do this, it is desirable to first sterilize the beads used.

Such beads can be easily sterilized by heat treatment, for example by treating them at temperatures on the order of 100° C. for several hours. This treatment removes the covalently bound proteins but leaves the silicone on the surface of the glass. The invention therefore also encompasses a method for restoring the bacteriostatic properties of glass microbeads, characterized in that microbeads sterilized by heat treatment are then treated by a method of covalently binding proteins as described above. shall be.

The present invention will be explained in more detail with reference to the following examples.

Example 1 Soda-alkali lime glass microbeads were sieved to remove beads smaller than 65 μm and larger than 106 μm. Average diameter (by weight) of selected beads 85μ
It was m.

The microbeads are mixed at a ratio of 0.1 head of silane per bead I Kf (this is about 100 silane per bead I Kf).
) in the form of an alcoholic solution at room temperature (T-
(glycidoxypropyl)trimethoxysilane (Union Carbide A187).

Lactoferrin was then covalently attached to the beads. Lactoferrin was attached to the epoxy group of the silane via its amino acid. This operation was carried out at room temperature by bringing the silanized beads into contact with a 10% titer aqueous solution of bovine lactoferrin, commercially available by Oleofina, at pH 4-5. This resulted in 0.01% by weight of active product based on the weight of the glass.

The microbeads were then gently heated, being careful not to exceed 60°C, and brought into contact with the silicone fluid. The silicone was Dow Corning's polydimethylsiloxane copolymer MDX 4-4159 containing amide 7 groups, which has a ratio of 0.2 CH/Kg of glass (
This corresponds to about 200 silane per kg of beads).

The bacteriostatic potency of these microbeads was the same as that of lactoferrin in unpassivated solution. For example, these microbeads were found to inhibit the growth of Niskerichia coli strains.

These microbeads were hydrophobic and could be stored, used, and manipulated without risk of aggregation.

Example 2 The same alkali lime glass microbeads as the microbeads in Example 1 were mixed with Union Carbide's (T-aminopropyl)trimethoxysilane Al1 in the form of an alcoholic solution.
00, silane o, i for glass l Kg
oC ratio (this is about 1 silane for glass l K7)
00WvI). The surface of the beads was then activated by reacting glutaraldehyde and silane in theoretical proportions at a pH below 6.5.

Lactoperoxidase was bound to the beads by contacting the beads with an aqueous solution of lactoperoxidase commercially available by Oleofina. This resulted in covalent bonding of 0.02% by weight of lactoperoxidase to the glass.

Next, the same silicone as in Example 1 was attached to the surface of the beads.

It was found that lactoperoxidase retained its enzymatic activity despite being covalently bound to glass. This enzyme activity was analyzed using the O-phenylene-diamine method to determine the 350% concentration of lactoperoxidase bound to glass.
A value of U/q was observed, whereas the same method used for the same amount of lactoperoxidase in solution form had an activity of about 400 υ/fng of the enzyme (U/■ is 1
is the unit of specific activity of a protein for 1 minute; one unit of activity is the unit of activity that produces an absorption increase of 0.1 at a wavelength of 490 nm in 1 minute at 37°C at pH 5 using HIO2 and 0-phenylene-diamine as substrates. )

For comparison, microbeads treated as described above but without silicone were placed in contact with the same Niskerichia coli strain. For the same amount of lactoperoxidase bound to the beads, the activity of the lactoperoxidase and silicone coated beads was ultimately the same as that of the non-siliconized beads. What was surprising here was that there was no interference between the presence of silicone and the activity of the passivated enzyme. The same result was obtained for other bacteria,
For example, it has also been found in Pseudomonas and Stachylococcus aureus.

Example 3 (T-glycidoxypropyl)trimethoxysilane was first coupled to the same glass microbeads as in Example 1 as described in Example 1, and 0.05% by weight of lactoperoxidase was directly attached. bound to microbeads. The same silicone as described in Examples 1 and 2 was deposited to complete the process.

Repeat the bacterial culture test of Example 2 and add 7g of beads per culture medium 11 to stop bacterial growth.
was found to be sufficient.

Claims (1)

[Scope of Claims] 1. Glass microbeads characterized in that they are coated with a protein that covalently binds to glass and imparts antibacterial properties to the beads. 2. The microbead according to claim 1, wherein the microbead has a non-porous surface. 3. The glass microbead according to claim 1 or 2, wherein the coating contains an enzyme. 4. The microbead according to claim 1 or 2, wherein the protein is selected from lactoferin and lactoperoxidase. 5. Claims 1 to 5, wherein the protein is present in a proportion of less than 0.1% by weight, preferably less than 0.05% by weight of the microbeads.
4. Microbeads according to any one of 4. 6. The microbead according to any one of claims 1 to 5, wherein the protein is bonded to glass via a silane coupling agent. 7. The microbead according to claim 6, wherein the protein is bonded to glass via silane and a second coupling agent. 8. The microbead according to claim 7, wherein the second coupling agent is glutaraldehyde. 9. Microbeads according to any one of claims 1 to 8, wherein they are hydrophobic. 10. Microbeads according to claim 9, wherein they carry a silicone coating. 11. Average diameter is 80-100 μm, preferably 85-100 μm
Microbeads according to any one of claims 1 to 10, having a diameter of between 90 μm. 12. Claims 1 to 1, wherein they are suspended in a fluidizing gas.
11. Microbeads according to any one of 11. 13. A bed for treating burn patients, comprising a fluidization device containing the microbeads according to claim 12. 14. A method for imparting bacteriostatic properties to glass microbeads, characterized in that a protein coating is covalently bonded to the glass microbeads. 15. The method of claim 14, wherein the beads are treated with a silane-based coupling agent before contacting the beads with the protein-containing medium. 16. The method of claim 15, wherein a second coupling agent is deposited on the beads subsequent to the silanization treatment. 17. The method of any of claims 14 to 16, wherein the protein is bound to the beads by bringing the beads into contact with a medium containing the protein having a pH of less than 7. 18. After the proteins have bound to the beads, they are brought into contact with a solution containing silicone.
7. The method described in any one of 7. 19. The method of claim 18, wherein the beads are sterilized by heat treatment before being coated with the protein. 20. A method for restoring the bacteriostatic properties of the glass microbeads used, which comprises treating the siliconized microbeads treated by the method according to claim 19 with the method according to any one of claims 14 to 17.