NO175854B - Glass microspheres with bacteriostatic properties and also bed for treating patients with burns, comprising a fluidization system. - Google Patents

Description

The present invention relates to glass microspheres with bacteriostatic properties, and their use in floating beds in hospitals intended for the treatment of patients with burns.

The bacteriostatic and bactericidal properties of a number of synthetic and 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 people with severe burns have been used in hospitals. These beds comprise fluidized bedding, i.e. a mattress or pillows consisting of particles brought to a fluidized state in a gas such as air and held in place under a gas permeable blanket. Sometimes the bed simply consists of a steel container containing fine, solid particles. The lower part includes a blower device that sucks air from the room through a filter. The air is compressed and heated to a temperature of 31-38°C. The air enters the layer of fine particles, e.g. of a thickness of 25 cm, and brings them to a fluidized state. The particles are trapped under a gas-permeable cloth produced, e.g. of polyester. All fluids drained from the patient can pass through such a cloth to form agglomerates with some particles, and such agglomerates are periodically removed by means of a sieve placed at the bottom of the container.

It is obvious that bedding of this type should not contain or promote the presence of microbial or bacterial strains. According to FR patent application 2,523,841, it has thus been proposed that particles of another material which have bactericidal properties are added to particles such as glass microspheres which make up the fluidized medium. The proposed particles are metal particles, such as calcium or magnesium or aluminum or bismuth or silicon particles. Apart from difficulties that may arise from the selection of the size of such particles to enable them to fluidize without segregation from the glass microspheres, it is obvious that a proposal of this type represents a definite risk in use. There is a risk of the particles igniting in the presence of moisture or 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 with a view to giving said bedding equipment bactericidal or bacteriostatic properties.

According to the present invention, glass microspheres are provided which are characterized by the fact that the spheres are coated with proteins which are bound covalently with a coupling agent to the glass and give the spheres bacteriostatic properties, and that the spheres are hydrophobic.

The present invention therefore represents an answer to the problem of giving bed equipment bacteriocidal or bacteriostatic properties because it enables single particles to be introduced into fluidized or floating beds in hospitals, namely glass microspheres which in themselves possess bacteriostatic properties. Furthermore, such particles can be recycled and regenerated.

Present microspheres to which proteins that give them a bacteriostatic ability are covalently bound, retain their properties over time, and these properties are maintained for several months before use if the beads are stored under good conditions, in dry, tight packaging, and they retain their bacteriostatic ability for a period that is compatible with their use in floating beds, i.e. for several days or even several weeks exposure to the air. This bacteriostatic ability is maintained when the balls are in a humid atmosphere, or when they are exposed to a moderate temperature (eg below 60 or 70°C). The bacteriostatic ability is preserved even when the balls are exposed in each other's surroundings to wear and tear during their handling and use. This is believed to be due to the covalent bond that occurs between the proteins and the glass. It has unexpectedly been found that despite the fact that said proteins are bound to the glass via a covalent bond, the proteins retain their bacteriostatic properties and are able to impart such properties to the microspheres that support them.

As already mentioned above, the microspheres possess bacteriostatic properties, and in many cases they are also bacteriocide, depending on the nature of the proteins and their concentration. For example, it is very easy to make them capable of killing or limiting the growth and reproduction of bacteria such as Escherichia coli, Salmonellae, Pseudo-monas, Legionellae and Staphylococcus aureus.

Present microspheres can be used in direct contact with the skin, e.g. in bandages, and in this case a special biodegradable glass will preferably be chosen. It is also possible to imagine binding to the microspheres of proteins that make them active, from a bactericidal point of view, in the digestive tract of humans and animals. It is also possible to use them to form a sterile medium that is not in contact with the body, as is the case for fluidized bed equipment. In consideration of the importance of this latter application, reference is made in particular to this in the present context, but it should be understood that the invention is not only limited to such use.

After the present microspheres have been in use for a certain period of time, it may be necessary or desirable to regenerate them for new use. It will e.g. it is usually necessary to change the balls in fluidized bed equipment for the treatment of patients with burns when a new patient is transferred to the bed. Used balls can be easily sterilized by heat treatment. They can e.g. processed at a temperature of approx. 100°C for a sufficiently long period of time. Such treatment also removes the protein coating from the balls, but a fresh coating of protein can easily be covalently bound to the balls so that they can be used again.

In the most preferred embodiments of the invention, said microspheres have a non-porous surface. This feature represents significant advantages from a hygienic point of view. Any body fluid, or other fluids, that come into contact with spheres that have porous surfaces can be easily adsorbed. Although this adsorbed material can be easily sterilized as mentioned above, it is very difficult to remove from porous beads so that it remains as a possible health hazard, even after sterilization and binding of bacteriostatic proteins to the beads. Should the bacteriostatic coating on such a porous sphere fail or fail, the adsorbed material can be a fertile microbiological breeding ground. This is not the case with spheres that have non-porous surfaces. Very little, if any, of the material of the type mentioned is found to be adsorbed, and what is adsorbed can easily be removed by sterilization so that it is possible to reuse the balls with much less risk of danger to the patient. Balls having non-porous surfaces have the additional advantage over balls with porous surfaces that they are generally easier to manufacture, and therefore less expensive.

The proteins bound to the glass microspheres are preferably enzymes. The choice of enzymes enables the growth or multiplication of bacteria to be suppressed or prevented in accordance with an overall selective, specific mechanism that enables the formation in situ of a self-contained entity harmful to bacteria. The preferred enzymes for use in connection with floating beds are peroxidases which catalyze the oxidation of various ions present in fluids such as plasma or serum, creating bactericidal elements.

Proteins such as transferrin or myeloperoxidase can be bound to the beads, but it is preferred to choose proteins such as lactoferrin or lactoperoxidase. Lactoferrin absorbs iron ions and thereby creates a medium that is unsuitable for bacterial growth. Lactoperoxidase, with an oxidizing agent such as hydrogen peroxide, provided metabolically or by the action of glucose oxidase on glucose (and SCN" ions form a medium containing OSCN ions which destroys bacteria.

Since the bacteriostatic effect of proteins is very effective, it is not necessary to completely coat the surfaces of the spheres with proteins. The proteins are preferably present in an amount of 0.1 wt-# in relation to the weight of the glass. An amount as small as 0.05 wt-# is effective, and it is often sufficient to use an amount of 0.02$.

As indicated above, a major advantage of the present microspheres lies in the fact that they retain their bacteriostatic or bactericidal properties despite mechanical and mechanical influences to which they may be subjected. This is probably due to the occurrence of a covalent bond between the proteins and the glass. To facilitate covalent attachment of the proteins to the glass, it is preferred to use a coupling agent which can create selective attachment sites for the reactive groups in the proteins at the glass surface. These reactive groups consist of amino acids. As a coupling agent it is possible to use an organic titanate, but it is preferred to choose a coupling agent of the silane type because the amount of silanes offers an extensive choice of substances that can react directly with amino acids.

As a variant, it may be preferred to bind the proteins using a silane compound and another coupling agent. In this case, the silane compound creates an aminated glass surface which is bound to the proteins via a "Michael" type linkage with glutaraldehyde, or of the amide type, e.g. with succinic anhydride, or of the azo type, e.g. with nitro-benzoyl chloride. It is also possible to form a thioester bond in the link chain, which has the advantage that it can be easily cleaved if it is desired to separate the beads from the proteins for recycling purposes. Coupling with glutaraldehyde is advantageous because it allows rapid binding of a variety of different proteins or different enzymes to the glass.

The present microspheres are preferably hydrophobic. This property enables them to retain their integrity in the presence of moisture or of various physiological fluids, and above all makes it possible to prevent them from agglomerating in the presence of these fluids. Present hydrophobic microspheres preferably owe their hydrophobic nature to the presence of a silicone coating. A silicone material is preferably chosen which polymerises at the free glass surface without being bound to it via covalent bonds. It can e.g. an amino group-containing polydimethylsiloxane copolymer such as the product Dow Corning MDX4-4159 is used, which polymerizes at room temperature or moderate temperature. A silicone material of this type is compatible with the presence of proteins and surprisingly does not modify or only slightly modifies the bacteriostatic activity of the microspheres compared to identical microspheres coated only with proteins.

Present glass microspheres are solid glass microspheres or hollow microspheres. For use in hospital floating beds, it is preferred that these microspheres have a relative density greater than 1 to facilitate patient support, although hollow microspheres have the advantage of requiring a smaller amount of fluidizing air, resulting in less out- drying of the fluidization atmosphere.

Microspheres are preferably chosen whose particle size distribution is narrow, which facilitates their fluidization without segregation. It is chosen e.g. glass microspheres whose diameter is between 65 and 110 micrometres. It is possible to use microspheres which have an average diameter between 80 and 100 micrometres, and preferably between 85 and 90 micrometres. Such spheres do not require too much energy for their fluidization, and are not able to pass through the network in covering materials normally used for floating beds in hospitals. For this particular application, solid glass microspheres or hollow microspheres can be selected.

The present invention thus also provides a bed for the treatment of patients with burns, comprising a fluidization system, characterized in that it contains present microspheres as defined above and which are suspended in a fluidizing gas.

By attaching a coating of proteins which are bound covalently to the glass for the production of the glass microspheres, a finely divided particulate material is obtained which can be easily fluidized, and which possesses and retains effective bacteriostatic properties. The microspheres are brought into contact with proteins, e.g. in suspension or in powder form.

The step of bringing the microspheres into contact with the proteins is preceded by a step of binding the coupling agent to the glass surface. The coupling agent is preferably a silane compound which is easily deposited on the glass surface from a solution, suspension or powder, at room temperature. It is e.g. it is also possible that amino or oxirane groups are grafted onto the glass surface which can react with the amino acids in the proteins.

In some cases, it may be useful to treat the glass with a different coupling agent. This is the case e.g. if the silane treatment of the glass forms amino groups at the glass surface. In this case, the protein is brought via a coupling of the "Michael" type with glutaraldehyde, or of the amide or azo type. In order to avoid denaturation of the proteins j, it is recommended to bind the proteins to the glass by bringing the latter into contact with a medium containing said proteins, in which the pH value does not exceed 7. A medium containing the proteins and in which the pH value is between 4 and 5, is preferably selected.

After said proteins have been bound to the glass, the microspheres are preferably brought into contact with a medium containing a silicone compound to deposit a silicone coating on the spheres. The spheres treated in this way are hydrophobic and have no tendency to agglomerate. The medium containing the silicone compound preferably has a pE value between 4 and 5. The deposition of silicone is carried out at room temperature or moderate temperature. In order not to denature the proteins, it is generally recommended to deposit and, where appropriate, polymerize the silicone material at a temperature that does not exceed 60-70°C.

When the microspheres have been used for a certain period of time, e.g. during a change of patients in a floating bed for the treatment of burns, it may prove necessary to regenerate the microspheres. To do this, it is desirable to first sterilize the used balls. Such balls can be easily sterilized by a heat treatment, e.g. by treating them at a temperature of 100°C for several hours. This treatment removes the covalently bound proteins, but retains the silicone material at the glass surface.

In the following, the invention will be explained in more detail with reference to the examples below.

Example 1

Massive alkali-lime glass microspheres are selected by sieving to remove spheres smaller than 65 micrometers and larger than 106 micrometers. The average diameter (calculated by weight) of the balls selected is 85 micrometers. The microspheres are treated with (gamma-glycidoxypropyl)trimethoxysilane (A 187 from Union Carbide) in alcoholic solution at room temperature, in an amount of 0.1 cm<3> silane per kg balls, which corresponds to approx. 100 mg silane per kg balls.

Lactoferrin is then covalently bound to the beads. The lactoferrin is bound via its amino acids to the epoxy groups in the silane. This operation takes place at room temperature by bringing the silane-treated balls into contact with a 10% aqueous solution of bovine lactoferrin (marketed by Oléofina) at a pH value between 4 and 5. Thus approx. 0.01 weight-$ active product calculated on the weight of the glass.

The microspheres are then gently heated, taking care not to exceed 60°C, and they are then brought into contact with a silicone liquid. The silicone chosen is an amino group-containing polydimethylsiloxane copolymer MDX4-4159 from Dow Corning, which is used in an amount of 0.2 cm<3> per kg of glass, which corresponds to approx. 200 mg silane per kg balls.

The bacteriostatic ability of these microspheres is identical to that of lactoferrin in solution, i.e. not immobilized. It is e.g. found that these microspheres inhibit the growth of an E. coli strain.

These microspheres are hydrophobic and can be stored, manipulated and used without any risk of agglomeration.

Example 2

Alkali-lime glass microspheres similar to those in Example 1 are treated using (gamma-aminopropyl)trimethoxysilane A1100 from Union Carbide in an alcoholic solution in an amount of 0.1 cm<3> silane per kg of glass, which corresponds to 100 mg of silane per kg of glass. The surface of the spheres is then activated by reacting the silane with glutaraldehyde at a pH value below 6.5 in stoichiometric quantities.

Lactoperoxidase is then bound to the beads by bringing them into contact with an aqueous solution of lactoperoxidase (marketed by Oléofina). 0^02 wt-# of lactoperoxidase, calculated on the glass, is then bound covalently. A silicone material identical to that in example 1 is then deposited on the surface of the spheres.

It was found that the lactoperoxidase retained its enzymatic activity despite its covalent binding to the glass. This enzymatic activity was analyzed by the o-phenylene-diamine method and a value of 350 U/mg of lactoperoxidase bound to the glass was observed, while the same method used on an equivalent amount of lactoperoxidase in solution had an activity of approx. 400 U/mg of enzyme (U/mg = unit of specific activity of the protein per mg; a unit of activity is the amount of enzyme which, within 1 min., gives an increase in absorbance at wavelength 490 nm of 0.1, at 37 °C and at pH 5, and with o-phenylenediamine and HgOg as substrate).

The bacteriostatic efficiency of the microspheres was also investigated. This bacteriostatic efficiency was analyzed with respect to an E. coli strain which was sensitive to the action of 0SCN~ions. The change with time of the optical density of such culture at wavelength 660 nm was investigated. An increase in optical density is evidence of bacterial growth. 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 magnitude of 100 after 6 hours at 37°C. In contrast, the optical density remains in the presence of 8 g per liter of spheres treated according to the present example (which corresponds to 500 U of lactoperoxidase per liter of culture medium), unchanged after 6 hours, and this demonstrates the blocking of bacterial growth.

For comparison, microspheres treated as described above, but not containing silicone, were contacted with the same E coli strain. For an identical amount of lactoperoxidase bound to the beads, it was found that the activity of the beads coated with lactoperoxidase and silicone is practically identical to that of the non-silicone treated beads. Thus, surprisingly, there is no interference between the activity of the immobilized enzyme and the presence of silicone material.

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

Example 3

First, (gamma-glycidoxypropyl)trimethoxysilane as described in Example 1 is bound to glass microspheres identical to those in Example 1, and 0.5 wt # of lactoperoxidase is then bound directly to the microspheres. The treatment is completed by depositing silicone identical to that indicated in examples 1 and '2. The test with cultivation of bacteria as in example 2 was repeated, and it was found that 7 g of balls per liter of culture medium was sufficient to block bacterial growth.

Claims (12)

1 Glass microspheres, characterized in that the spheres are coated with proteins that are bound covalently with a coupling agent to the glass and give the spheres bacteriostatic properties, and that the spheres are hydrophobic. 2. Microspheres according to claim 1, characterized in that they have a non-porous surface. 3. Microspheres according to claim 1 or 2, characterized in that the coating comprises an enzyme. 4. Microspheres according to claim 1 or 2, characterized in that the proteins are selected from lactoferrin or lactoperoxidase. 5 . Microspheres according to any one of claims 1-4, characterized in that the proteins are present in an amount of less than 0.1 wt-# calculated on the microspheres, and preferably less than 0.05 wt-#. 6. Microspheres according to any one of claims 1-5, characterized in that the proteins are bound to the glass via a coupling agent of the silane type. 7. Microspheres according to claim 6, characterized in that the proteins are bound to the glass via a silane and another coupling agent. 8. Microspheres according to claim 7, characterized in that the second coupling agent is glutaraldehyde. 9. Microspheres according to claim 1. characterized in that they carry a silicone coating. 10. Microspheres according to any one of claims 1-9, characterized in that their average diameter is between 80 and 100 micrometers, and preferably between 85 and 90 micrometers. 11. Microspheres according to any one of claims 1-10, characterized in that they are suspended in a fluidizing gas. 12. Bed for treating patients with burns, comprising a fluidization system, characterized in that it contains microspheres according to claim 11.