FI95458B - Glass microbeads - Google Patents

Description

95-56 GLASS HIKROHELMEI.

This invention relates to lssisis microbeads having becquerio-eteaty properties, and to their use as hospital lei.iu mattresses for the treatment of burn patients.

Bacterioacetates and bactericidal properties among synthetic and natural products have long been studied, and some of these products form part of a 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. Accordingly, in FR patent application 2,523,841, it has been proposed that particles of a second substance having bacterioacetate properties be added to particles such as glass microbeads which form a fluidized medium. The proposed particles are metal particles, such as calcium or 2 95-5b '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 clear 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 such bed area pasting with 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 with a covalent linker 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 the beads in question, 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 giving 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 .1 ill i nilti nmt 3 9 5 * 5 fa ·

Overall, it has been unexpectedly found that despite the fact that these proteins are bound to glass by a covalent bond, the proteins retain their bacteriostatic properties and are able to impart these properties to the microbeads that underlie them.

As already stated above, microspheres 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 multiplication, such as Escherischla coll, Salmonella, Pseudomonas, Leqionellae and Staphylococcus aureus.

The microbeads according to the invention can be used in direct contact with the skin, for example in waterways, in which 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 the latter use, the present invention refers specifically to s'i, but it is to be understood that the present invention is not 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 95-56

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 in 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 allows for the reduction or inhibition of bacterial growth or multiplication, overall by a selective specific mechanism,! which allows the bacteria born in the situation to be harmful. Preferred enzymes for the fluidized bed application support are peroxidases, which catalyze the oxidation of various ions present in fluids such as plasma and serum, creating bacterial entities.

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 an oxidizing agent such as hydrogen peroxide (obtained metabolically or) I l I l I Still I I! · »I 95 '5f; glucose oxidase to glucose) and SCN® ions in a medium containing OSCN- ions that 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 microspheres according to 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, yelane generates an aminated glass surface associated with proteins by a "Michael" -type coupling, with glutaraldehyde, or an amide-type coupling, e.g., succinic anhydride, or an azotypic coupling, e.g., nitrobenzoyl chloride. It is also possible to generate a thioester crosslink in the linker chain, which has the advantage that it can be easily cleaved if it is desired to separate the beads from the proteins for recycling. Coupling with glutaraldehyde is advantageous because it allows a large number of proteins or various enzymes to be rapidly bound to glass.

6 9 5 * 5 fc

The microspheres according to the invention are 6 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. 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 fluidizing air, resulting in less dehydration.

Preferably, microbeads with a narrow particle size distribution are selected, which facilitates the fluidization of the coals 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.

il I i «: t nid h tm.

The present invention also encompasses microbeads having bacteriostatic properties suspended in fluidizing gas and comprising a bed for the treatment of burn patients comprising a fluidizing system comprising microbeads as defined above.

The lens beads of the invention have been given bacteriostatic properties, with the microbeads having a protein coating covalently bound to the glass. Such a structure has the advantage of obtaining a finely divided particulate material which can be easily fluidized and which has and retains effective bacteriostatic properties. The protein coating is applied to the microbeads by contact with the protein population, 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. That is the case. for example, if silanization of 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.

95 * 56

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 doping of the silicone is performed at room temperature or at a reasonable temperature. To avoid denaturation of the proteins, it is generally recommended that the silicone be blended and suitably polymerized at a temperature not higher than 60-70 ° C.

After 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 microbeads can be maintained by treating the microbeads heat-sterilized as described above, which covalently binds the proteins.

The present invention will be 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 im i ane 11 <tft: i 95 * 5b

The microgelat is 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 films. Lacto-ferrin is bound via its amino acids to the silane groups of the silane. 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 contacted with silicone tea. 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 * · ·

The same age glass microbeads as in Example 1 are treated using Union Carbide (gamma-aminopropyl) trimethoxysilane A1100 in an alcoholic 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 "5έ ·

The lactoperoxidase is then bound to the beads by so-called contact with an aqueous solution of lactoperoxidase marketed as 0166. 0.02% by weight of lactoperoxidase 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 lactoperoxidase 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 by the same method an equivalent amount of lactoperoxidase 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 bacteriostatic 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 beads coated with an identical amount of bead-bound lactoperoxidase and lactoperoxidase and ailicone is seemingly identical compared to silicone-free beads A <tt f Rilli I M # 1 i <11 9 5'-5 b. 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 precipitation of 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. Glaucoma beads, characterized in that the beads are coated with mad proteins, which are bound covalently to the glass by a coupling agent and give the beads bacterioethaic properties and that the beads 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 4. Microbeads according to claim Teller 2, characterized in that the proteins are selected from the lactoferrin and lactoperoxidase. Microbeads according to the nigot 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. A microbead 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 bound 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. 9. Microbeads according to claim 1, characterized in that they carry a silicone coating. 10. Microbeads according to the figure of claims 1 to 9, characterized in that their average diameter is between 80 and 100 micrometers, and preferably between 85 and 90 micrometers. k, 9 5 11. Microprocessors according to one of Claims 1 to 10, characterized in that they are suspended in a fluidization gas. 5 12. Bed for the treatment of burn patients, comprising a fluid therapy, syst, characterized in that it contains micropeads as claimed in claim 11.