AU611936B2 - Glass microbeads having bacteriostatic properties and process for manufacturing such microbeads - Google Patents

Description

n cll~--*-~qR__CY11 rra~~ glll*---~~ar I-i COMMONWEALTH OF AUSTRALIA Patents Act 19 11936 C O M P L E T E S P E.C I FI--C-A T I O N

(ORIGINAL)

Application Number Lodged Complete Specification Lodged Accepted Published S Priority 7 September 1987 Related Art 0r Name of Applicant :GLAVERBEL 0 9 Address of Applicant chaussee de la Hulpe, 166 B 1170 Brussels- Belgium o Actual Inventor/s :Marcel Delzant 0 0 Address for Service F.B. RICE CO., Patent Attorneys, 28A Montague Street, Balmain N.S.W. 2041 Complete Specification for the invention entitled: GLASS MICROBEADS HAVING BACTERIOSTATIC PROPERTIES AND PROCESS FOR MANUFACTURING SUCH MICROBEADS The following statement is a full description of this invention including the best method of performing it known to us:- I i i 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 burn 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 individuals suffering from severe burns have been used in hospitals. These beds comprise fluidized .i Jo 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 o« particles. The base comprises a blower which sucks air from the room through a I 0. filter. This air is compressed and heated to a temperature of the order of 31 to 0 15 38°C. The air enters a layer of fine particles, for example approximately 25 cm 0 °0 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, I; o*9 and such agglomerates are periodically removed by means of a sieve placed at the "0 20 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 FR 2.523.841, 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 i; l' bedding with bactericidal or bacteriostatic properties.

According to the present invention, glass microbeads are characterized in that the beads are coated with proteins wbi h are bound covalently to the glass and endow the beads with bacteriostatic properties.

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 fluidized beds, namely glass microbeads which in themselves possess bacteriostatic properties. Furthermore, such particles can be recycled and regenerated.

c 10 The microbeads according to the invention, to which proteins which Io provide them with a bacteriostatic power are bound covalently, retain their properties with the passage of time; these properties are maintained for several months 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 o a damp atmosphere or when they are subjected to a moderate temperature (for example below 60 or 70 0 The bacteriostatic power is preserved even when the beads are subjected among themselves to abrasion during their handling and use.

o 20 This is thought to be due to the covalent bond existing between the proteins and 0 the 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 0 bacteriostatic properties and are capable of imparting such properties to the microbeads which support them.

S 25 As already stated above, the microbeads possess bacteriostatic i properties and, in many cases, they are also bactericidal, depending on the nature I of the proteins and their concentration. By way of example, it is very easy to render them capable of killing or limiting the growth and multiplication of 1 bacteria such as Escherichia coli, Salnonellae, Pseudomonas, 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 envisage binding to the microbeads proteins which make them active, from the bactericidal 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 I--q I ~Yll I PIY-~~ L1 3.

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 microbeads according to the invention have been in use for a certain period of time, it may be necessary or desirable to regenerate them for reuse. For example it will generally be necessary to change the beads 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 100°C for a sufficient length of time.

Such a treatment also removes the protein coating from the beads, but a fresh 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, 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 oo above, it is extremely difficult to remove from porous beads, so that it remains as a o oi possible health hazard, even after sterilization and bonding of bacteriostatic proteins to the beads. If the bacteriostatic coating of such a porous bead should 4. o 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 So i 0 o 25 hazard to the patient. Beads having non-porous surfaces have the further advantage over beads with porous surfaces that they are in general easier and therefore less expensive to produce.

d 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 mechanism enabling an entity detrimental to bacteria to be created in situ. The preferred enzymes for application to fluidized 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 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 4 I4 4.

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 completely to coat the surfaces of the beads with proteins. Preferably, 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 small as 0.05% by weight is effective, and it is often sufficient to use a quantity of 0.02%.

As stated above, a great advantage of the microbeads according to the invention resides in the fact that the latter retain their bacteriostatic or o1 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 s5 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 oOo coupling agent, since the range of silanes offers a wide choice of substances Scapable of reacting directly with amino acids.

By way of a variant, it may be preferable to bind the proteins by o 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 azo type, for example with nitrobenzoyl chloride. It 0, 0, is also possible to create a thioester bond in the coupling chain, which has the advantage that it can be readily cleaved if it is desired to separate the beads from i 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 hydrophobic. This property enables them to keep their integrity in the presence of moisture or of various physiological fluids, and makes it possible, above all, to prevent them from agglomerating in the presence of these fluids. The hydrophobic microbeads according to the invention preferably owe their hydrophobic nature to the presence of a silicone coating. A silicone which 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, L; I which polymerizes at room temperature or moderate temperature, may be used.

A silicone of this kind is compatible with the presence of proteins and, surprisingly, does not modify or only negligibly modifies the bacteriostatic activity of the microbeads with respect to identical microbeads coated only with proteins.

The glass microbeads according to the invention are solid glass microbeads or hollow microbeads. 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 smaller quantity of fluidization air, resulting in less 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 4 4 15 and 100 micrometres, and preferably between 85 and 90 micrometres. Such beads do not require too much energy for their fluidization, and are not capable of 0o passing through the meshes of the cloths normally used for hospital fluidized beds.

For this particular application, solid glass microbeads or hollow microbeads may be selected.

o 20 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 microbeads 0 as defined above.

o The present invention also relates to a process for endowing glass microbeads with bacteriostatic properties, characterized in that a coating of proteins that are bound covalently to the glass is attached to these microbeads. A process of this kind has the advantage of obtaining a finely divided particulate material 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.

xl 6.

In some cases, it may be useful to treat the glass with a second coupling agent. This is the case, for example, 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 the azo type. In order to avoid denaturation of the proteins, it is recommended 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 excecd 7. A medium containing the proteins in which the pH is between 4 and 5 is preferably selected.

O 10 Preferably, after the said proteins have been bound to the glass, the microbeads are brought into contact with a medium containing a silicone, in order ;o 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 carried out at room temperature or moderate temperature. In general, in order not to denature the proteins, it is recommended to deposit and, where appropriate, to polymerize the silicone at a temperature not exceeding 60 to 70 0

C.

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 burn 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 temperature of the order of 100°C for several hours. This treatment removes the covalently bound proteins but retains the silicone at the surface of the glass. Accordingly, the invention also encompasses a process for restoring the bacteriostatic properties of glass i microbeads, characterized in that the microbeads which have been sterilized by a heat 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 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 diameter (by weight) of the beads selected is 85 micrometres.

The microbeads are treated with (gammaglycidoxypropyl)trimethoxysilane (A 187 of Union Carbide) in alcoholic solution at room temperature, in the proportion of 0.1 cc of silane per kilogramme of 1 A i-~ii~o O o 0 00 0 4O o r 7.

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 place at room temperature, by bringing the silane-treated beads into contact with a 10% strength aqueous solution of bovine lactoferrin marketed by Ol6ofina, at a pH 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 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 kilogramme of glass, which corresponds to approximately 200 mg of silane per kilogramme of beads.

15 The bacteriostatic power of these microbeads is identical to that of lactoferrin in solution, that is to say not immobilized. It has been found, for example, 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.

20 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 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 Ol6ofina.

0.02% by weight of lactoperoxidase with respect to the glass is thereby bound 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. 'his enzymatic activity was assayed by the o-phenylene-diamine method, and a value of 350 U/mg of lacto peroxidase 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 mg; one unit of 8.

activity is the quantity of enzyme which produces, in one minute, an increase in absorbance at wavelength 490nm of 0.1, at 37°C and at pH 5, and with ophenylene-diamine and H 2 0 2 as substrate).

The bacteriostatic power of the microbeads was also examined. This 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 corresponding to a multiplication of the initial value by a factor of the order of 100 after 6 hours at 37 0 C. In contrast, in the presence of 8 g 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 15 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 S, that, for an identical quantity of lactoperoxidase 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 the activity of the immobilized enzyme and the presence of the silicone.

Similar results are found with other bacteria such as Pseudomonas and Staphylococcus aureus.

,Example 3 S In the first place, (gamma-glycidoxypropyl)trimethoxysilane is bound S 25 as described in Example 1 to glass microbeads identical to those of Example 1, and 0.05% by weight of lactoperoxidase is then bound directly to the microbeads. "he treatment is completed 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.

Claims (8)

1. 9. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:- 1. Glass microbeads, characterized in that the beads are coated with proteins which are bound covalently to the glass and endow the beads with bacteriostatic properties. 2. Microbeads according to claim 1, wherein the said microbeads have a non-porous surface. 3. Glass microbeads according to claim 1 or 2, wherein the coating comprises an enzyme. 4. Microbeads according to claim 1 or 2, wherein the said proteins are chosen from lactoferrin and lactoperoxidase. 5. Microbeads according to one of claims 1 to 4, wherein the said proteins are present in the proportion of less than 0.1% by weight of the microbeads, and preferably less than 0.05,,o by weight. S* 6. Microbeads according to one of claims 1 to 5, wherein the said proteins are bound to the glass via a silane-type coupling agent. 7. Microbeads according to claim 6, wherein the said proteins are bound to the glass via a silane and a second coupling agent. 4 Microbeads according to claim 7, wherein the second coupling agent is glutaraldehyde. 9. Microbeads according to one of claims 1 to 8, wherein they are hydrophobic. Microbeads according to claim 9, wherein they bear a silicone coating. o o 11. Microbeads according to one of claims 1 to 10, wherein their average diameter lies between 80 and 100 micrometres, and preferably between and 90 micrometres.
2. 12. Microbeads according to one of claims 1 to 11, wherein they are suspended in a fluidization gas.
3. 13. Bed for the treatment of burn patients, comprising a fluidization system, characterized in that it contains microbeads according to claim 12.
4. 14. Process for imparting bacteriostatic properties to glass microbeads, characterized in that a coating of proteins is covalently bound to the glass microbeads. Process according to claim 14, wherein the said beads are treated by means of a silane type coupling agent prior to their being brought into contact with a medium containing proteins. I I I I I e' I
5. 16. Process according to claim 15, wherein the silanization treatment is followed by the deposition of a second coupling agent on the beads.
6. 17. Process according to one of claims 14 to 16, wherein the said proteins are bound to the beads by bringing the latter into contact with a medium containing the said proteins in which the pH is below 7.
7. 18. Process according to one of claims 14 to 17, wherein, after the said proteins have been bound to the beads, they are brought into contact with a solution containing a silicone.
8. 19. Process according to claim 18, wherein the beads are sterilized by o1 a heat treatment before coating with protein. Process for restoring bacteriostatic properties of used glass microbeads, wherein siliconized microbeads treated by a process according to claim 19 are then treated by a process according to one of claims 14 to 17. Dated this 30th day of August 1988 GLAVERBEL Patent Attorneys for the Applicant F.B. RICE CO. !1 00 0 0, 00 0 003 00 0U O 00 0xaar o 03C