FI95458C - Glass microbeads - Google Patents

Description

95 * 56 GLASS MICROBeads.

This invention relates to glass microspheres having bacteriostatic properties, and to their use as hospital bed mattresses for the treatment of burn patients.

Bacteriostatics and bactericidal properties Among synthetic and natural products have long been studied, And some of these products form part of the traditional pharmacopoeia, such as antibiotics and disinfectants ·

For some years, special beds have been used in hospitals to treat people suffering from severe burns. These beds comprise a fluidized lower part, such as a mattress or pillows, consisting of particles placed in a fluidized state in a gas, such as air, and held in place under a gas-permeable fabric. Sometimes the bed simply comprises a steel tank containing fine solid particles. The bottom comprises a fan that sucks air from the room through a filter. This air is compressed and heated to a temperature of, for example, 31-38 ° C. The air passes into a layer of fine particles, for example about 25 cm thick, and places them in a fluidized state. The particles remain under a gas-permeable fabric made of, for example, polyester. Liquids leaking from the patient can pass through such a fabric and form aylomers with some of the particles, and these agglomerates are removed periodically by means of a screen placed at the bottom of the tank.

It is obvious that such a bedding medium should not contain or promote the presence of microbial or bacterial strains. Thus, in FR patent application 2,523,841, it has been proposed to add particles of another substance with bacteriostatic properties to particles such as glass microspheres which form a fluidized medium. The proposed particles are metal particles, such as calcium or 2 95 * -56 'magnesium or aluminum or bismuth, or silicon particles. In addition to the difficulties that may arise in selecting the size of these particles in order to be able to float indistinguishably from the glass beads, it is obvious that a proposal of this kind involves a certain risk in use. In the presence of moisture or in a hot place, there is a risk of ignition, or in the case of calcium, already at room temperature. Therefore, it is desirable to find other means that are simpler and safer for obtaining such a bed area having bactericidal or bacteriostatic properties.

According to the present invention, glass microbeads are characterized in that the beads are coated with proteins covalently bound to the glass by a coupling agent and impart bacteriostatic properties to the beads, and that the microbeads are hydrophobic.

The present invention thus provides an answer to the problem of imparting bacteriocidal or bacteriostatic properties to such beads, since it makes it possible to use particles of the simple type to be added to hospital Lei-jupa mattress beds, namely glass microspheres which themselves have bacteriostatic properties. In addition, these particles can be recycled and regenerated.

. The microbeads of the invention, to which the proteins that give them their bacteriostatic ability are covalently bound, retain their properties over time; these properties are maintained for several months before use if the beds are stored in good conditions, in a litter, in a sealed package, and retain their bacteriostatic ability over time suitable for use in fluidized bed beds, even for several days or even several weeks in contact with air. This bacteriostatic ability is maintained when the beads are in a humid atmosphere or when kept at a reasonable temperature (e.g., 60 or 70 ° C). The bacteriostatic ability is maintained even when the beads are exposed to wear during handling and use. This is thought to be due to the hard-bonded bond between the proteins and the glass. Overall, it has unexpectedly been found that, despite the fact that the proteins in question have been fenced in glass by means of a hard-bonded bond, the proteins retain their bacteriostatic properties and are able to impart these properties to microbeads, which are their basis.

As already stated above, freshwater beads have bacteriostatic properties and in many cases are also bactericidal, depending on the nature of the proteins and their concentration. For example, it is easy to make them capable of killing the following bacteria or restricting their growth and replication, such as Escherischia coli, Salmonella, Pseudomonas, Legionellae and Staphylococcus aureus.

The microbeads according to the invention can be used in direct contact with the skin, for example in the skin, and in this case a special biodegradable glass is preferably chosen. It is also possible to take into account that proteins which make them active, bactericidal, in the digestive tract of humans and animals can be bound to the microbeads. It is also possible to use them to create a sterile medium that is not in contact with the body, as is the case in a fluidized bed. Emphasizing the importance of this latter use, the present invention specifically refers to it, but it is to be understood that the present invention is not * · 4 ': exclusively limited to this use.

After the microbeads of the invention have been used for a certain period of time, it may be necessary or desirable to regenerate them for new use. For example, it is usually necessary to change the beads of the fluidized bed for the treatment of burn patients when a new patient is transferred to the bed. Used beds can be easily sterilized by heat treatment. They can be treated, for example, at a temperature of about 100 ° C for a sufficiently long time. Such treatment also removes the protein coating from the beads, but the fresh protein coating can be easily covalently bound to the beads so that they can be reused.

4 95 * 5fc '

In the most preferred embodiments of the invention, said microbeads have a non-porous surface. Adopting this fact brings considerable benefits in terms of hygiene. Any substance or other liquid that comes into contact with the beads »having a porous surface can be easily adsorbed. Although this adsorbed material can be easily sterilized, as mentioned above, it is extremely difficult to remove it from the porous beads, so that it remains a potential health hazard, even after sterilization and binding of bacteriostatic proteins to the beads. If the bacteriostatic coating of such a porous bead fails, the adsorbed material may be a fertile microbiological culture medium. This is not the case with beads with a non-porous surface. Little, if any, of such material is found to be adsorbed, and what is adsorbed is easily removed during sterilization, so that it is possible to reuse the beads and there is a lower risk of endangering the patient. Non-porous surface beads have the advantage over porous surface beads that they are generally easier and therefore cheaper to manufacture.

Preferably, the proteins that bind to the glass microbeads are enzymes. The choice of enzymes makes it possible to reduce or inhibit the growth or multiplication of the bacteria, all in all, according to a selective specific mechanism: which allows a whole that is harmful to the bacteria born in the situation. Preferred enzymes for fluidized bed application are peroxidases, which catalyze the oxidation of various ions present in fluids such as plasma and serum, generating whole bacterial mouths.

Proteins such as transferrin or myeloperoxidase may be bound to the beads, but it is preferred to select proteins such as lactoferrin or lactoperoxidase. Lactoferrin takes up iron ions and thus creates a medium that is unsuitable for bacterial growth. Lactoperoxidase forms a medium with an oxidizing agent such as hydrogen peroxide (obtained metabolically or by the action of glucose oxidase on glucose) and SCN "ions / which contains OSCN 'ions which kill bacteria.

Because the bacteriostatic action of proteins is very efficient, it is not necessary to completely cover the surfaces of the beads with proteins. Preferably, the proteins are present in an amount of less than 0.1% by weight based on the weight of the glass. An amount of less than 0.05% by weight is effective and it is often sufficient to use an amount of 0.02%.

As stated above, the great advantage of the capped microspheres of the invention is that they retain their bacteriostatic or bactericidal properties despite the mechanical and thermal stresses to which they are subjected. This is probably related to the existence of a covalent bond between proteins and glass. To facilitate covalent attachment of proteins to glass, it is preferred to use a coupling agent that is capable of creating preferred attachment sites on the surface of the glass for the reactive groups of the proteins. These reactive groups consist of amino acids. It is possible to use an organic titanate as the coupling agent, but it is preferable to choose a silane-type coupling agent because the range of silanes offers a wide range of substances capable of reacting directly with amino acids.

: Alternatively, it is preferred to bind the proteins with a silane and another coupling agent. In this case, the silane creates an aminated glass surface associated with proteins by a "Michael" -type coupling with glutaraldehyde, or an amide-type coupling, for example succinic anhydride, or an azotypic coupling, for example nitrobenzoyl chloride. the advantage that it can be easily cleaved if it is desired to separate the beads from the proteins for recirculation.The coupling with glutaraldehyde is advantageous because it allows a large number of proteins or various enzymes to be rapidly bound to the glass.

6 95 * 5b

The microbeads according to the invention are 6 or more hydrophobic. This property allows them to maintain their integrity in the presence of moisture or various physiological fluids and, above all, helps to prevent them from agglomerating in the presence of these fluids. The hydrophobic nature of the hydrophobic microbeads of the invention is due to the silicone coating present. Preferably, a silicone is selected which polymerizes on the free surface of the glass without binding to it by covalent bonds. For example, a polydimethylsiloxane copolymer containing a smino group can be used, such as Dow Corning MDX4-4159, which polymerizes at room temperature or at a reasonable temperature. Such silicone is compatible with the presence of proteins and not surprisingly alters or alters only a negligible amount of bacteriostatic activity of microspheres compared to identical microbeads coated with proteins only.

The glass microbeads according to the invention are solid glass microbeads or hollow microbeads. For use in hospital fluidized bed beds, it is preferred that the relative density of these microbeads be greater than 1, making it easier to support the patient, although hollow microbeads have the advantage of requiring a smaller volume of fluid. air, which results in less drying of the fluidizing atmosphere.

Preferably, microbeads with a narrow particle size distribution are selected, which facilitates their fluidization without agglomeration. For example, glass beads with a diameter between '65 and 110 micrometers are selected. It is possible to use microbeads with an average diameter of between 80 and 100 micrometers, and preferably between 85 and 90 micrometers. Such beads do not require too much energy for fluidization and are unable to pass through the loops of fabrics normally used in hospital fluidized bed mattresses. Solid glass microbeads or hollow microbeads can be selected for this special purpose.

The present invention also encompasses microbeads having bacteriostatic properties and suspended in le- lt. III 11 1: 1 t ¢ 1 7 95 * 5b. and comprises a bed for the treatment of burn patients comprising a fluidization system containing microbeads as defined above.

The lens beads of the invention have been given bacteriostatic properties, with the microbeads having a protein coating bound to the glass covalently. The TB-like structure has the advantage of obtaining a finely divided particulate blue material which can be easily fluidized and which has and retains effective bacterioetatic properties. The protein coating is provided on the microspheres in contact with the proteins, for example in the form of a suspension or powder.

The step of contacting the microbeads with the proteins is preferably preceded by binding of the coupling agent to the surface of the glass. The coupling agent is preferably yelane, which readily precipitates on the glass surface from a solution, suspension or powder at room temperature. It is also possible to attach, for example, amino or oxirane groups to the surface of the glass with a side chain, which can react with the amino acids of the proteins.

In some cases, it may be useful to treat the glass with another coupling agent. This is the case, for example, if the silanization of the glass generates amino groups on the surface of the glass. In this case, the protein is bound by a "Michael" -type coupling with glutaraldehyde, or by an amide-type or azotypic coupling. To avoid denaturation of the proteins, it is recommended to perform the binding of the proteins to the glass by contacting the glass with a medium containing said proteins, wherein the pH does not exceed 7. Preferably, a protein-containing medium having a pH between 4-5 is selected.

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After these proteins are bound to the glass, the microbeads are preferably contacted with a medium containing silicone to cause the silicone coating to precipitate on the surface of the beads. Beads treated in this way are hydrophobic and do not tend to agglomerate. The pH of the silicone-containing medium is preferably between 4 and 5. The precipitation of silicone is carried out at room temperature or at a reasonable temperature. To avoid denaturation of the proteins, it is generally recommended that the silicone be precipitated and suitably polymerized at a temperature not exceeding 60-70 ° C.

When the microbeads have been used for a certain period of time, for example the time between patient changes in a fluidized bed for burn cases, it may be necessary to regenerate the microbeads. To accomplish this, it is desirable to first sterilize the spent beads. These beads can be easily sterilized by heat treatment, for example, at 100 ° C for several hours. This treatment removes covalently bound proteins but retains silicone on the glass surface. However, the bacteriostatic properties of the microspheres can be maintained by treating the microbeads sterilized by hot hands with the above as described above, which covalently binds the proteins.

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

'Example 1

Solid alkali-lime glass microbeads are selected by screening out beads smaller than 65 micrometers and larger than 106 micrometers. The average diameter (by weight) of the selected beads is 85 micrometers.

il lii j liiti I t t Iti: l 9 9 5 ^ 5 b

The microbeads are treated with (gammaglycidoxypropyl) triraethoxysilane (A 187 from Union Carbide) in an alcoholic solution at room temperature, in a ratio of 1 ml of silane per kilogram of beads, corresponding to approximately 100 mg of silane per kilogram of beads.

The lactoferrin is then covalently bound to the beads. Lacto-ferrin is bound to the epoxy groups of the silane via its amino acids. This operation is performed at room temperature by contacting the silane-treated beads with a 10% concentrated aqueous solution of bovine lactoferrin, marketed as 0-14ofina, at a pH between 4-5. Here, there is approximately 0.01% by weight of active product relative to the weight of the glass.

The microbeads are then gently heated, taking care not to exceed 60 ° C, and brought into contact with the silicone fluid. The silicone selected is Dow Corning's amino group-containing polydimethylsiloxane copolymer MDX4-4159, used in an amount of 0.2 ml per kilogram of glass, corresponding to approximately 200 mg of silane per kilogram of beads.

The bacteriostatic ability of these microspheres is identical to that of lactoferrin in solution, meaning not immobilized. For example, it has been found that these microbeads inhibit the growth of an E. coli strain.

These microbeads are hydrophobic, can be stored, handled and used without the risk of agglomeration.

Example 2

Time-lapse microbeads similar to Example 1 are treated using Union Carbide (gararo-aminopropyl) -trimethoxysilane A1100 alcohol solution in an amount of 0.1 ml of silane per kilogram of glass, corresponding to approximately 100 mg of silane per kilogram 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.

10 95''5i-

The lactoperocidase is then bound to the films by contacting them with an aqueous solution of lactoperoxidase marketed as 0166. 0.02% by weight of lactopsroxidase relative to the glass thus binds covalently. Silicone identical to the silicone of Example 1 is then deposited on the surface of the beads.

It was found that lactoperokidase retains its enzymatic activity despite covalent binding to glass. This enzymatic activity was determined by the o-phenylenediamine method and found to be 350 U / mg lactoperoxidase bound to glass, whereas when used in the same method an equivalent amount of lactopsroxidase in solution gave an activity of about 400 u / mg enzyme (U / mg protein per milligram of specific activity per unit one unit of activity is the amount of enzyme which produces an increase in absorbance of 0,1, at 490 nm, at 37 ° C and pH 5, in one minute, and o-phenylenediamine and H2O2 as nutrient solution).

The bacteriostatic ability of the microspheres was also studied. This bacterioetatic ability was determined for an E. coli strain sensitive to OSCN- ions. The change in optical density of such a culture over time at 660 nm was studied. An increase in optical density indicates the growth of bacteria. In the absence of lactoperoxidase, the optical density of the control increases, corresponding to an amplification of the initial value of about 100 after 6 hours at 37 ° C. In contrast, in the presence of Θ g per liter of beads treated according to this example (corresponding to 500 U lactoperoxidase per liter of culture medium), the optical density is unchanged after 6 hours, indicating inhibition of bacterial growth.

For comparison, microbeads treated as described above but free of silicone were contacted with the same E. coli strain. It was found that the activity of the beads coated with an identical amount of herniated lactoperoxidase and lactone oxidase is seemingly identical compared to the siliconized beads. Thus, surprisingly, there is no difference here between the activity of the immobilized enzyme and the presence of yesterday.

similar results are obtained with other bacteria such as Pseudomonas and Staphylococcus aureus.

Example 3

First, (gammaglycidoxypropyl) trimethoxysilane is bound 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.

The treatment is supplemented by doping a silicone identical to the silicone shown in Examples 1 and 2.

The bacterial culture experiment of Example 2 was repeated, and it was found that 7 g of beads per liter of culture medium was sufficient to inhibit bacterial growth.

Claims (12)

1. Glaemic beads, characterized in that the beads are coated with proteins that are covalently bound to the glass with a coupling agent and give the beads bacterioethatic properties, and that the microbeads are hydrophobic. 2. Microbeads according to claim 1, characterized in that the microbeads have a non-porous surface. Glass microbeads according to claim 1 or 2, characterized in that the coating contains an enzyme. 10 Microbeads according to claim 1 or 2, characterized in that the proteins are selected from the lactoferrin and lactoperoxidase. Microbeads according to any one of claims 1 to 4, characterized in that the proteins are present in a proportion of less than 0.1% by weight of the microbeads, and preferably less than 0.05% by weight. 6. Microbeads according to any one of claims 1 to 5, characterized in that the proteins are bound to the glass via a silane-type coupling agent. 7. Micro beads according to claim 6, characterized in that the proteins are bonded to the glass via a screen and a second coupling agent. Microbeads according to claim 7, characterized in that the second coupling agent is glutaraldehyde. '30 9. Microbeads according to claim 1, characterized in that they wore a silicone cover. Microbeads according to any one of claims 1 to 9, characterized in that their average diameter is between 80 and 100 micrometers, and preferably between 85 and 90 micrometers. 14 9 5 ^ 5b Micropubles according to any one of claims 1 to 10, characterized in that they are suspended in a fluidized gas. 5 Bed for treating burned patients, containing a fluidisation cysteine, characterized in that it contains micropeads as claimed in claim 11.