Specifically antibacterial coating of surfaces with bacteriophages for medical applications

Abstract

Bacteriophages are suitable for the targeted antibacterial treatment of surfaces with active substances that are specifically active against bacteria. These are introduced into polymer coatings which are bioresorbable and thus allow for a continuous release of the bacteriophages. The bacteriophages are introduced into the polymer solution after prior encapsulation with a reversible hydrocolloid. Hydrolysed collagen, which forms a gel under native conditions and is also reversible, was used as the hydrocolloid. The coating solution is applied by means of dip-coating methods. The coated surfaces have an antibacterial effect and are not cytotoxic.

Description

Specifically antibacterial coating of surfaces for medical technology applications

[0001] The patent describes a composition for a coating for the antibacterial treatment of surfaces with bacteriophages, which is antibacterially effective and bioresorbable. The core of the invention is the incorporation of the bacteriophages in a bioactive form into a polymer solution and their permanent release through encapsulation by means of a reversible hydrogel. The antibacterial coating is intended in particular for invasive medical devices such as catheters, implants, etc. [State of the art]

[0002] Antibacterial finishes on products, especially medical devices, are often achieved using antibiotics, organic active ingredients (e.g. quaternary ammonium compounds) or inorganic substances, especially silver compounds.

[0003] For example, patent US10589003B2 describes an antibacterial coating of medical surfaces with the antibiotics minocycline and rifampicin by impregnation with an antibiotic solution and subsequent removal of the solvent by tempering.

[0004] Another example of antibacterial equipment for medical surfaces is patent US7354605B2, which describes an antibacterial equipment or coating of metal ions encapsulated in a hydrophilic polymer.

[0005] However, these often have the disadvantage that they do not have a specifically antibacterial effect, but rather their mechanism of action is based on general cell damage caused by the binding of the released silver ions or active ingredients. This non-specific cell damage affects both prokaryotic and eukaryotic cells. Therefore, such antibacterial medical devices often also exhibit cytotoxic properties.

[0006] Furthermore, the use of antibiotics, disinfectants, or silver compounds carries the risk of bacterial resistance developing. The development of bacterial resistance, particularly to antibiotics, is well-documented.

[0007] Alternative antibacterial agents are known, including enzymes (e.g., lysozyme), antimicrobial peptides, and bacteriophages. Due to their thermal instability, these bioactive substances are often applied to surfaces via covalent bonding or coating. Disadvantages of covalent bonding to the surface include inhibition of migration and the resulting limited surface activity, as well as the potential inactivation of the antibacterial mechanism through chemical coupling.

[0008] US10478401B2 and JP2023513919A describe how bioactive antibacterial finishes are implemented in coatings, mostly by encapsulation in alginate gels. However, the release from the polysaccharide is limited according to the size of the antibacterial additives. Furthermore, alginate gel is not reversible under physiological conditions, so release of larger molecules is not possible under physiological conditions. The coating itself is also often implemented using hydrogels.

[0009] The production of an antibacterial coating with biomolecules and polymers that are only soluble in denaturing solvents is not possible due to the use of native active ingredients. [Objective of the invention]

[0010] The object of the invention is to provide a specifically antibacterial polymer coating for medical devices that has a targeted antibacterial effect and whose active ingredients are continuously released.

[0011] To overcome the disadvantage of the non-specific effect of previously used antibacterial agents, specifically antibacterial components are to be used; in the case of the present invention, these are bacteriophages.

[0012] Furthermore, the disadvantage of the reduced release of antibacterial components in coatings with covalently bonded components must be overcome, and the preservation of the native forms of the specifically antibacterial components must be technically solved. This also applies to polymer coatings.

[0013] The core objective of the invention was therefore to develop a polymer coating that a) obtains a native form of the bacteriophages in a solvent-based polymer coating solution, b) enables a general release of the bacteriophages from the coating, c) enables a time-delayed release of the phages from the coating and d) represents a stable coating.

[0014] The problem is solved by producing a solution of bacteriophages and a bioresorbable polymer, which can be used as an antibacterial coating.

[0015] Bacteriophages are a group of viruses that infect specific bacteria, replicate within them, and are released after lysis of the bacterial cells. They combine the advantage of a specific antibacterial mechanism of action with biocompatibility with human cells.

[0016] The release of bacteriophages from the coating, which can migrate from the surface into the surrounding tissue in a gradient, is achieved by embedding the bacteriophages in a bioresorbable polymer, such as polyhydroxyalkanoate (PHA) or polylactic acid (PLA). These bioresorbable polymers are not water-soluble but primarily soluble in chloroform. To protect the native bacteriophages from denaturation by the solvent, they are first encapsulated in a water-swellable, chloroform-insoluble hydrocolloid. Hydrolyzed collagen was used for encapsulating the bacteriophages as the only hydrocolloid that gels reversibly under physiological conditions. This allows for both the release of the bacteriophages from the hydrogel and their embedding in a chloroform-containing polymer solution.

[0017] Specifically, the task is solved by the following steps: a) Suspending bacteriophages in collagen solution b) Producing gel microstructures by spraying the bacteriophages / collagen suspension from a) into a phosphate buffer solution tempered to 1-5°C and separating the coagulated gel microstructures, or alternatively, producing gel microstructures by freezing the bacteriophages / collagen suspension from a) and subsequent cryogenic milling c) Preparation of a solution of a bioresorbable polymer in an organic solvent; optionally, aqueous polyvinyl alcohol solution and lecithin can be added and stirred to form a suspension. d) Addition of the gel microstructures from one of the two variants from b) to the solution / suspension from c). e) Dip coating of the body to be coated in the solution / suspension from d) and subsequent drying.

[0018] Bioresorbable within the meaning of the invention means that the material is broken down in the body by means of hydrolysis and / or enzymatically. The time until complete degradation depends on the polymer used.

[0019] Hydrocolloids or hydrogels within the meaning of the invention are polysaccharides or proteins that form a colloidal solution (gel) in water. Examples include alginate and hydrolyzed collagen, in particular gelatin.

[0020] Bacteriophages within the meaning of the invention are viruses that infect bacteria in a species-specific manner, replicate within the bacterial cell and are released again by lysis.

[0021] The bacteriophages used as antibacterial agents in medical devices are specifically effective against human pathogenic bacteria, including, preferentially, phages against Enterobacterales (including Citrobacter spp., Enterobacter spp., Escherichia coli, Klebsiella spp., Morganella morganii, Proteus spp., Plesiomonas shigelloides, Providencia rettgeri, Salmonella typhi, other Salmonella spp., Serratia marcescens, Shigella spp., Yersinia enterocolitica, Y. pestis), and Gram-positive cocci (including Staphylococcus aureus, S. epidermidis, Enterococcus faecalis, E. faecium, Streptococcus agalactiae, S. bovis, S. pneumoniae, S. pyogenes, S. mutans, S. mitis). S. salivarius, S. sanguis, S. anginosus, S. milleri, S. constellatus, Gemella morbillorum), nonfermenters - non-Enterobacterales (including Acinetobacter calcoaceticus, Elizabethkingia meningoseptica, Pseudomonas aeruginosa, P. alcaligenes, other Pseudomonas spp Stenotrophomonas maltophilia) and others .

[0022] Examples include the phages Citrobacter phage vB_CfrM-Bl; Escherichia phages; Pseudomonas phages; Staphylococcus phage EBHT MB.LM vB_SauM_155Rindl, vB_SepP_UKE3; be Klebsiella phages.

[0023] The encapsulation of the bacteriophages is achieved by introducing a bacteriophage suspension into a liquid hydrocolloid whose sol-gel transition is reversible under physiological conditions (37 °C, physiological solution). Hydrolyzed collagen is suitable for this purpose. The collagen is dissolved in phosphate-buffered saline solution with heating, preferably at a concentration of 0.1 to 0.5 g / ml. The solution is cooled to approximately 45 °C, and 1 / 20 to 1 / 2 volume of a bacteriophage suspension with a titer of at least 5 × 10⁻⁵ is added while stirring. 7 PFU / ml is added. The suspension is then formed into gel microcapsules by spraying into cold phosphate-buffered saline (PBS).

[0024] Alternatively, the bacteriophage-collagen suspension of dissolved hydrolyzed collagen and bacteriophages is transferred to sterile glass containers and stored at room temperature until solidified. The gel mass is then coarsely crushed mechanically and further finely ground using a cryogenic mill.

[0025] The coating solution is prepared by dissolving a bioresorbable polymer, preferably a polyhydroxyalkanoate (PHA) or polylactide (PLA), in a solvent, preferably chloroform. From the PHA, preferably polyhydroxybutyrate homopolymer (PHB), or Poly(hydroxybutyrate-co-hydroxyvalerate) copolymers (PHBV) are used to prepare a polymer solution with a concentration in the range of 0.03–0.10 g / ml. For polylactide, concentrations in the range of 0.05–0.15 g / ml are used. To optimize the viscosity and adhesive properties of the PHA solutions, polyvinyl alcohol (PVA) was added as a film former. For this purpose, an approximately 20% aqueous PVA solution was added to the PHA solution in a volume ratio of 1:2 v / v. Lecithin was also added as an emulsifier at a concentration of approximately 10 mg / ml. No further optimization of the PLA coating solution was necessary.

[0026] The microencapsulated bacteriophages were added by pipetting or weighing the gel into the coating solution in a ratio of 1:10 v / v or w / v while stirring at 100 rpm immediately before the coating process.

[0027] The surface coating was carried out using a dip-coating process, in which, for example, catheters, preferably made of thermoplastic polymers, including thermoplastic polyurethane (TPU) or polyethylene (PE), or polysiloxanes (silicone), were sealed at the ends, briefly dipped into the coating solution and then dried on a device at room temperature.

[0028] Bacteriophages, which specifically infect human pathogenic bacteria, are suitable for the antibacterial treatment of surfaces. This antibacterial treatment with bacteriophages can be achieved using coating processes (impregnation, dip-coating). The coating can consist of a polymer, preferably a bioresorbable polymer. Due to the solubility of most bioresorbable polymers in chloroform, the bacteriophages must be encapsulated in a microcapsule / protective shell that allows for the preservation and release of the native bacteriophages. Hydrolyzed collagen is particularly suitable for this purpose. The quantity of bacteriophages is at least 5 × 10⁻⁵. 7 PFU / ml .

[0029] The antibacterial coating is primarily intended for the application to invasive medical surfaces, particularly catheters and implants, that are temporarily or permanently inserted into the body and carry the risk of bacterial infection, either upon insertion of the medical device or postoperatively. An example is the antibacterial coating of a bladder catheter with Escherichia bacteriophages, such as T4 or a mixture of different Escherichia phages, for the prevention of catheter-associated urinary tract infections. Other applications include intravascular catheters, such as peripherally inserted intravascular or central venous catheters, which can be coated with a bacteriophage consisting of phage t>3 against coagulase-negative staphylococci and Staphylococcus aureus to prevent catheter-associated infections. Brief description of the illustrations: Figure 1: TPU catheter with coating made of PVA: hydrolyzed collagen : PHV : lecithin Figure 2: TPU catheter with PLA coating containing 1 / 10 volume of hydrolyzed collagen Figure 3: Agar diffusion test of coated catheter samples. PK1: Positive control 1 = TPU coated with hydrolyzed collagen containing 0.5 mg / ml gentamicin; PK2: Positive control 2 = TPU coated with hydrolyzed collagen containing 1 / 10 vol. Serratia phage kappa strain suspension; NK1: Negative control 1 = TPU with hydrolyzed collagen; NK2: Negative control 2 = TPU with PVA; NK3: Negative control 3 = TPU with PVA: Hydrolyzed collagen: PHV ENMAT Y1000; PI: Sample 1 = TPU coated with hydrolyzed collagen containing bacteriophage Serratia phage kappa, PVA, and PHV; P2: TPU with a multilayer structure consisting of 1. hydrolyzed collagen containing bacteriophage Serratia phage kappa, 2. PVA, and 3. PVA and PHV. Figure 4: Agar diffusion test of coated catheter samples. PK1: Positive control 1 = TPU coated with hydrolyzed collagen containing 0.5 mg / ml gentamicin; PK2: Positive control 2 = hydrolyzed collagen with 1 / 10 vol. Serratia phage kappa strain suspension; NK: Negative control = TPU with PLA; PI: Sample 1 = TPU coated with hydrolyzed collagen containing the bacteriophage Serratia phage kappa [Examples] Materials: - Bacteriophages: Pseudomonas phage phi6 DSM 21518, Serratia phage kappa DSM 14097, Leibniz Institute DSMZ, DE - Poly(hydroxybutyrate-co-hydroxyvalerate) copolymer (PHBV), ENMAT™ PHBV resin Y1000P, Specific gravity = 1.25, DSC melting point = 175-180 °C, TianAn Biopolymer / Helian Polymers, NL - Hydrolyzed collagen / gelatin, CAS No. 9000-70-8, 140 Bloom, 20-30 mesh, protein content 83-91%, Carl Roth GmbH + Co. KG, DE - Ix Phosphate-buffered saline solution (PBS) , prepared from Ix DPBS w / o Ca w / o Mg, 500 ml, Biowest SAS, F - Lecithin / Phosphatidylcholine, CAS No. 8002-43-5, Carl Roth GmbH + Co. KG, DE - Chloroform CHC13, CAS No. 67-66-3, Th. Geyer GmbH & Co . KG Methods: Antibacterial efficacy was determined using an agar plate diffusion test according to DIN EN ISO 20645 and ISO 22196. In vitro cytotoxicity was determined according to DIN EN ISO 10993-5. Preparation of the phage collagen solution: The bacteriophages [min. 5 • 10 7 [PFU / ml] are suspended in hydrolyzed collagen tempered to 45 °C. The hydrolyzed collagen used is gelatin [0.17 g / ml, Bloom value 120], which is first swollen in phosphate-buffered saline solution and heated to liquefying at 80 °C for at least 1 hour. It is then tempered to 45 °C. One-tenth of the volume of the bacterial suspension is added to the liquefied, hydrolyzed collagen by pipetting. Example 1: The bioresorbable polymer PHBV ENMAT™ Y1000P is dissolved in chloroform at a concentration of 0.043 g / ml with stirring at room temperature. The polymer solution is mixed 2:1 (v / v) with an aqueous polyvinyl alcohol (PVA) solution MOWIOL 8-44 at a concentration of 0.133 g / ml, for example, 10 ml of PHBV solution with 5 ml of PVA solution. The immiscible polymer solutions are emulsified by adding 1 / 3 volume of a lecithin solution in chloroform at a concentration of 0.05 g / ml. Following this example, this would be 5 ml of lecithin solution. Approximately 1 / 10 volume of the bacteriophage-containing gel microstructures is added to the polymer mixture with a spatula while stirring at room temperature. This corresponds to approximately 3 g of the microcapsules. The surfaces are coated by dip coating with the polymer solution, whereby the bodies to be coated are immersed in the polymer solution for 10 seconds, removed and then dried at room temperature for 24 hours. Example 2: As an alternative to underwater spraying, the bacteriophage-containing microcapsules can be produced by cryo-milling. For this, the hydrolyzed collagen is stored at 4 °C after adding the bacteriophage suspension until solidification and then ground into a powder using cryo-milling (CryoMill). This powder is then added to 1 g / 10 ml of polymer solution and used for dip coating as described above. Example 3: As an alternative to PHA, the bioresorbable polymer polylactic acid (PLA) can be used as a coating polymer. For this, 11 g of PLA are dissolved in 100 ml of CHCl3 while stirring at room temperature. 1 g of hydrolyzed collagen microstructures or the cross-milled powder can then be added directly to this polymer solution and used for dip coating.

Claims

Patent claims:

1. A method for producing an antibacterial coating, characterized by the following steps: a. Suspending bacteriophages in collagen solution b. Producing gel microstructures by spraying the bacteriophages / collagen suspension from a) into a phosphate buffer solution tempered to 1-5°C and separating the coagulated gel microstructures, or alternatively, producing gel microstructures by freezing the bacteriophages / collagen suspension from a) and subsequent cryogenic milling c. Preparation of a solution of a bioresorbable polymer in an organic solvent, d. Addition of the gel microstructures from one of the two variants from b) to the solution / suspension from c) e. Immersion coating of the body to be coated in the solution / suspension from d) and subsequent drying .

2. Method according to claim 1, characterized in that in step c) aqueous polyvinyl alcohol solution and lecithin are additionally added and stirred to form a suspension.

3. Method according to claim 1 or 2, characterized in that the collagen is hydrolyzed collagen / gelatin.

4. A method according to one or more of claims 1 to 3, characterized in that the bacteriophages are specifically effective against human pathogenic bacteria, preferably phages against Enterobacterales, in particular against Citrobacter spp., Enterobacter spp., Escherichia coli, Klebsiella spp., Morganella morganii, Proteus spp., Plesiomonas shigelloides, Providencia rettgeri, Salmonella typhi, other Salmonella spp., Serratia marcescens, Shigella spp., Yersinia enterocolitica, Y. pestis, gram-positive cocci, in particular Staphylococcus aureus, S. epidermidis, Enterococcus faecalis, E. faecium, Streptococcus agalactiae, S. bovis, S. pneumoniae, S. pyogenes. S. mutans, S. mitis, S. salivarius, S. sanguis, S. anginosus, S. milleri, S. constellatus, Gemella morbillorum, nonfermenter.

5. Method according to one or more of claims 1 to 4, characterized in that the phages are in particular Citrobacter phage vB_CfrM-Bl ; Escherichia phages; Pseudomonas phages; Staphylococcus phage EBHT, MRLM, vB_SauM_155Rindl , vB_SepP_UKE3 ; Klebsiella phages .

6. Method according to one or more of claims 1 to 5, characterized in that the bioresorbable polymers are approved for medical use and are preferably selected from polyhydroxyalkanoate, polylactide, polyglycolic acids, polycaprolactone, or blends thereof.

7. Method according to one or more of claims 1 to 6, characterized in that the polyvinyl alcohol has a molar mass of at least 31,000 g / mol and a degree of hydrolysis of at least 88%.

8. Method according to one or more of claims 2 to 7, characterized in that the lecithin is obtained from soybeans and consists of at least 97% phospholipids.

9. Antibacterial coating with bacteriophages as an active component, characterized in that it is produced according to a method according to one or more of claims 1 to 8.

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