WO2019003090A1 - ANTIBACTERIAL COATING - Google Patents

Abstract

The present invention relates to a coating comprising metallic or magnetic nanoparticles functionalized and linked to bacteriophages, proteins or peptides with bacterial action derived from them. The antibacterial coating inhibits the colonization and growth of bio-dandruff-forming bacteria on surfaces and medical devices.

Description

 ANTIBACTERIAL COVERING

FIELD OF THE INVENTION The invention is part of the field of nanobiotechnology, particularly in products with antibacterial activity that can be used to coat surfaces and inhibit the colonization and growth of bacteria forming biofilms. BACKGROUND OF THE INVENTION

Since 2001, the World Health Organization (WHO) has called for the search of new antimicrobial alternatives, being the bacteriophage therapy or the experimentation with enzymes derived from them, one of the most studied alternatives. The fields with a high potential for the use of bacteriophages are: phage typing; phagotherapy; the decontamination of food; disinfection of medical instruments; the prevention of biofilm formation on surfaces; bacterial detection; the vehicles to deliver drugs; and molecular biology. The prevention of the formation of biofilms has become a problem of great importance in several sectors such as health, agroindustry, water, among others. Several strategies have been identified for the use of bacteriophages against bacterial films, including prevention by blocking the development of the biofilm (biofilms) and the elimination of an existing biofilm.

Himba et al. documented the use of phage to prevent the development of Listeria monocytogenes biofilms on a stainless steel surface using a phage solution with agitation. After 6 hours of treatment, the decrease in in vivo bioluminescence of the bacterial film formed on the steel sheet by the bacteriophages was observed. Likewise, they observed the prevention in the development of the film of L. monocytogenes on the steel sheets in bacterial cultures inoculated with the phage (Hibma AM, Jassim SAA, Griffiths MW. of L-forms of Listeria monocytogenes with bred bacteriophage. Int Journal Food Microbio .. 1997 [cited 2017 May 10]; 34 (34): 197-207. Available from: htt: // page s. cs .wisc.edu / -athula / yohani / Hibma.pdf]). Similar studies have been developed for the control of bacterial films of different species, among which are Staphylococcus epidermidis (Curtin JJ, Donlan RM.Using bacteriophages to reduce formation of catheter-associated biofilms by Staphylococcus epidermidis.Antimicrob Agents Chemother [Internet]. 2006 [cited 2017 May 11]; 50 (4): 1268-75, available at: http://www.ncbi.nlm.nih.gov/pubmed/16569839), Pseudomonas aeruginosa (Ahiwale S, Tamboli N, Thorat K , Kulkarni R, Ackermann H, Kapadnis B. In Vitro Management of Hospital Pseudomonas aeruginosa Biofilm Using Indi genous T7-Like Lytic Phage, Curr Microbiol [Internet], 2011 [cited 2017 May 11]; 62 (2): 335-40. Available at: http://link.springer.com/10.1007/s00284-010-9710-6) and Staphylococcus aureus because they are the most frequent causes of hospital infections (Parasion S, Kwiatek M, Gryko R, Mizak L, Malm A Bacteriophages as an alternative strategy for fighting biofilm development, Polish J Microbiol 2014; 63 (2): 137-45).

A potential use of bacteriophages is to prevent the formation of bacterial films on the surfaces of catheters and medical devices, although some variables arise, such as the infectivity and stability of the phage, the ability of a particular material to retain the phage and phage stability in the presence of body fluid compounds. In addition, the careful selection of phage cocktails, the optimization of the matrix and the validation of in vitro methods and in animal models are very important in the evaluation of the usefulness of bacteriophages in the fight against the formation of bacterial films (González S, Fernández L, Campelo AB, Gutiérrez D, Martínez B, Rodríguez A, et al The behavior of Staphylococcus aureus dual-species biofilms treated with bacteriophage philPLA-RODI depends on the accompanying microorganism Appl Environ Microbiol [Internet] 2016 AEM .02821-16 Available at: http://aem.asm.org/lookup/doi/10.1128/AEM.02821- 16). Hajar Maleki et al. describe the synthesis of magnetic nanoparticles (NPs) of superparamagnetic iron oxide (SPION). The nanoparticles can be functionalized with thiocarbamate groups and coupled with broad-spectrum antibacterial conjugated antibacterial peptides (AMP-CM) effective against intracellular pathogens (eg causing meningitis and tuberculosis) and infections associated with biomaterials (Hajar Malehi et al.) High antimicrobial activity and low human cell cytotoxicity of core-shell magnetic nanoparticles functionalized with an antimicrobial peptide ACS Appl. Mater. Interfaces, 2016, 8 (18), pp 1 1366-1 1378). US 20100291537 discloses devices to be employed as methods of treatment or in vivo or in vitro detection methods comprising phago-nanoparticle assemblies comprised of nanoparticles (size 2 to 500 nanometers) of Au, Ag, Pt, Ti, Al, Yes, Ge, Cu, Cr, W, Fe, or oxides thereof, linked with one or more phage particles (filaments), Escherichia coli plasmids, peptides, pyrrole, imidazole, histidine, cysteine or tryptophan. The phago- nanoparticle assembly can also include other agents, including therapeutic agents.

Scibilia et al. reveals a self-assembly approach for the construction of hybrid nanostructured networks of diverse applications in the biomedical field, consisting of clones of phage MI 3 P9b, specific for Pseudomonas aeruginosa, directly assembled with silver nanoparticles (AgNPs) obtained by pulsed laser ablation. The assembly of the networks is controlled by electrostatic interactions between the major capsid proteins of the pVIII phage and the AgNPs. (Scibilia et al., Self-assembly of silver nanoparticles and bacteriophage, Sensing and Bio-Sensing Research (2016), Article in Press.)

Taking into account bacterial film removal strategies or the prevention of their formation using phage solutions, there is a need to develop new products that inhibit bacterial growth and prevent the formation of bacterial films, reducing the possibility of generating new strains of bacteria with resistance to antibiotics or disinfectants., especially by high prevalence of multiresistant bacteria that are added in these films.

The bacteria in these films share and exchange genetic material, making the emergence of strains resistant to conventional antibiotics very easy. The use of bacteriophages and proteins derived from these allow to achieve a lower risk of developing resistance and increasing specificity. The phages evolve naturally as the bacteria change, ensuring, within the dynamics of infection, that the phage finds a way to infect and lyse its host.

BRIEF DESCRIPTION OF THE INVENTION

The invention relates to a coating comprising metallic and / or magnetic nanoparticles functionalized and linked with bacteriophages, proteins or peptides with antibacterial action derived therefrom. The coating allows to inhibit colonization and growth of bio-film forming bacteria.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1: Size distribution of gold nanoparticles on a silicon surface by atomic force microscopy.

FIG. 2: Morphology of modified Ag nanoparticles, a) UV-VIS spectrum of Ag nanoparticles b) TEM image of Ag nanoparticles c) Analysis of the frequency of size distribution of Ag nanoparticles d) Synthesis of nanoparticles of Ag Ag by laser induction with 50mW, 363, 8nm, for 10 seconds.

FIG. 3: Plaques of bacteriophages of E. coli using the double agar technique. DETAILED DESCRIPTION

The invention corresponds to an antibacterial coating that is formed by metallic or magnetic nanoparticles functionalized and linked with bacteriophages, also called phages, highly specific against bacteria resistant to antibiotics and biofilm formers. In addition to phages, metallic or magnetic nanoparticles can be fused with proteins or peptides with antimicrobial action.

The antibacterial coating according to the invention corresponds to a dispersion or suspension of functionalized metallic and / or magnetic nanoparticles in an aqueous medium containing bacteriophages, proteins or antibacterial peptides derived from phages. The aqueous medium can be distilled water or a buffer solution (e.g. TRIS, phosphate buffer, citrate buffer) with pH between 6.0 and 8.0. For purposes of the present invention, the functionalized metal nanoparticles are defined as nanoparticles of metals such as silver, gold, titanium, copper, iron, aluminum, platinum, tungsten, among others, with a particle size between 1 and 100 nanometers, to which they are bound by covalent bonds, hydroxyl, amino, carboxyl and thiol groups. Likewise, the functionalized magnetic nanoparticles are defined as nanoparticles of magnetite, maghemite, nickel, cobalt, among others, with a particle size between 1 and 100 nanometers, which have, bound by covalent bonds, hydroxyl, amino, carboxyl and thiol.

The functionalized metallic or magnetic nanoparticles are linked either with one or several types of bacteriophages or with proteins or peptides of antibacterial action coming from them. If the functionalized nanoparticle binds with bacteriophages, it does so through electrostatic interactions, whereas if the functionalized nanoparticle binds with proteins or peptides derived from phages, it does so covalently. It is important to determine the Z potential of the bacteriophages, proteins and peptides with antibacterial action in order to anchor them to the nanoparticles and determine exactly with which ligand the nanoparticle will be functionalized. To determine if the ligand is anchored chemically with the nanoparticle, characterization is necessary through spectroscopic techniques such as Raman or Infrared spectroscopy.

Bacteriophages (phages) are highly specific viruses that infect and kill bacteria. These viruses are found in each environment where their bacterial host is found (e.g., in aquatic, terrestrial environments, among others). Currently, more than 5500 different bacteriophages have been structurally described. Since its discovery, its use in the clinical field for the treatment of infectious diseases has been proposed, thus giving rise to phagotherapy, based on the bactericidal activity of these viruses (Ackermann HW, 2007. 5500 Phages examined in the electron microscope, Arch. Virol 152: 227-243).

Understanding phage biology is essential to maximize its use as growth inhibitors of biofilms formed by antibiotic resistant bacteria, since not all phages kill their host. Therefore, the multiplication process of the phages must be known, which can be of two types, lytic or lysogenic. Next, the stages of infection are described: a) Adsorption or binding to the host cell: Bacteria have specific receptors for the viruses that infect them, therefore the phages have host specificity and are capable of attacking only one type of host. bacteria, even differentiate them within the same species. b) Penetration, rupture of the membranes and bacterial walls, and nucleic acid in the cytoplasm of the parasitized cell: Adsorption and penetration can be of different modalities depending on the type of virus, being the most known case that of bacteriophages; After being fixed to the bacterial wall with the help of caudal fibers and the basal plate, they nail its tubular axis and inject its DNA. In many cases the bacteriophage uses proteins (eg enzymes) that break the bacterial membranes, these having the potential to be used in the coating of the present invention. Once the genomic material is inside of the bacterium, depending on the nature of the phage, the lysogenic cycle and / or the lytic cycle may occur.

The lysogenic cycle occurs when the viral nucleic acid does not express its genes, is integrated into the genome of the bacteria or is free as a plasmid. Both genomes are replicated together. The phage remains in the form of a prophage and the cell that houses it remains as a lysogenic cell. This process means an alteration, by genetic enrichment, of the lysogenic cell. The profane remains latent in the bacteria and is not activated unless the bacterium undergoes drastic changes that alter its DNA. When the prophecy is activated, it goes through a lytic cycle.

In the lytic cycle viral nucleic acid seizes cellular metabolism, directing it towards the manufacture of viral components: copies of viral nucleic acids, transcription of the message from its genome to mRNA and translation of this to proteins of the capsid and viral enzymes. These components accumulate in different parts of the infected cell. c) Assembly: When there is enough of these viral molecules, the nucleic acid is folded and introduced into the capsid, forming large amounts of virions. d) Release: Virions are released from the cell by different procedures, the most frequent being the lysis or disintegration of the membrane of the infected cell. In this phase of the lithic cycle are the other group of enzymes with potential for the coating, these are the endolysins together with holins and spanins.

The bacteriophages, their proteins, enzymes and antibacterial peptides fused with the functionalized metal nanoparticles of the coating of the invention, inhibit the formation of bacterial films of the genera Staphylococcus sp., Steptococcus sp., Pseudomonas sp, Kelbsiella sp., Salmonella sp., Eschericia coli, among others. In a embodiment of the invention, bacteriophages are selected from bacteriophages of

Klebsiella pneumoniae, Klebsiella oxytoca, Staphylococcus aureus and Escherichia coli.

In one embodiment of the invention, the concentration of the bacteriophages in the coating is between 1X10 ⁷ and 1X10 ¹⁴ PFU / mL plate forming units, preferably between 1X10 ¹⁰ and 1X10 ¹² , while the concentration of proteins and peptides with antibacterial action in the coating it is between 0.01 μg / mL and 100 μg / mL, preferably between 0.1 μg / mL and 20 μg / mL. The proteins (enzymes) derived from bacteriophages, which bind to the metallic or magnetic nanoparticles in the coating of the present invention, are selected from endolysins and depolymerases. Despolymerases are the proteins responsible for the first steps of viral infection, responsible for the degradation of the bacterial envelope, while the enzymes responsible for bacterial lysis are the endolysins, holins, and spanins (Bacteriophages and Phage-Derived Proteins). - Application Approaches, O'Flaherty S, Ross RP, Coffey A. Bacteriophage and Their Lysins for Elimination of Infectious Bacteria, FEMS Microbiol Rev. 2009; 33: 801- 819). Enzymes of polysaccharide-degrading phages are proteins, associated with virions, used to enzymatically degrade the capsular (eg alginate, hyaluronan, polysialic acid, amylovorane) or structural polysaccharides (eg, lipopolysaccharides and peptidoglycans) of the bacteria. The ability of phages to overcome these structures through these enzymes with depolymerizing activity allows these viruses to infect encapsulated bacteria that present high levels of multi-resistance (http://www.who.int/mediacentre/news/releases/2017/ bacterium- antibiotics-needed / en /).

The peptides that bind to the metallic or magnetic nanoparticles in the coating of the present invention are selected from peptides corresponding to the CHAP domains of endolysins. A common catalytic domain called CHAP has been described (by its English name cysteine-and-histidine-dependent aminohidrolase / peptidase), which presents hydrolase activity of peptidoglycan (PGH). CHAP has been characterized and tested in vivo using in vitro and in vivo models (Sanz-Gaitero et al., Crystal structure of the lytic CHAPK domain of the endolysin LysK from Staphylococcus aureus bacteriophage K. Virology Journal 2014. 11: 133).

The following Examples illustrate the invention, without the inventive concept being restricted thereto.

EXAMPLE 1. Synthesis of metal nanoparticles of Au.

In a vessel 50 mL of H [AuC] (0.01% w / v) were bubbled with nitrogen gas. In another container, a 1% (w / v) solution of sodium citrate was also bubbled with nitrogen gas and quickly the two solutions were taken to the incubator for 15 minutes to stabilize them.

Then a volume of 1.0 mL of the citrate solution was taken, it was added to the solution of H [AuC14]. Then the mixture was left to rest for 12 to 24 hours until the color was red to violet. The volume of added citrate can be varied to obtain different sizes of nanoparticles. The intensity of the coloration of the solution may be indicative of the average size of the nanoparticles.

EXAMPLE 2. Synthesis of metal nanoparticles of Ag.

The silver nanoparticle growth solution was prepared by the basic method described in the publications: Y. Sun .; B. Mayers .; Y. Xia. Transform on of silver nanospheres into nanobelts and triangular nanoplates through a thermal process. Nano Letters, 3 (5): 675-679, 2003; and M. Maillard .; H. Pinray .; B. Louis. Silver nanodisk growth by surface plasmon enhancedphotoreduction of adsorbed [ag + J. Nano Letters, 3 (11): 161 1-1615, 2003., which are incorporated in their entirety as a reference. A solution of silver nitrate (100 mL, 0.1 mM) and trisodium citrate (1 mL, 0.5 mM) was added with stirring and 0.5 to 0.7 mL of NaBH4 (10 mM) was added dropwise. ). An absorption maximum was observed in the 398-401 nm range that increased in intensity with each drop added. The addition of NaBFLt solution was stopped when the maximum absorption did not increase further.

EXAMPLE 3. Synthesis of magnetite magnetic nanoparticles.

The magnetite magnetic nanoparticles were obtained following the methodology of Subbiahdoss et al, 2012, by the polyol method (Subbiahdoss G. Magnetic targeting of surface-modified superparamagnetic ironoxide nanoparticles yields antibacterial efficacy against biofilmsof gentamicin-resistant staphylococci.) Acta biomaterialia 8 (6) : 2047-55). Aqueous solutions of FeCl ₂ * 4H ₂ 0 and FeCl ₃ * H ₂ 0 were mixed in diethylene glycol. The mixture was heated to 170 ° C for a period of 15 minutes, then NaOH was added and after 1 hour the magnetite nanoparticles were obtained, which were collected using a magnet and rinsed with an HNO ₃ solution.

EXAMPLE 4. Characterization of the metallic nanoparticles The characterization of the metallic nanoparticles obtained according to Examples 1 and 2 can be carried out with the help of a "Dynamic Light Scatterin" or more conventional forms such as the UV-Vis spectrum for the identification of the size of the metal nanoparticles. the nanoparticles through the resonance plasmon. Atomic force microscopy (AFM) is another alternative technique to verify the size of nanoparticles. In FIG. 1 shows the distribution of gold nanoparticle sizes (average of 12 ± 2nm) on a silicon surface.

EXAMPLE 5. Modification of the surface (functionalization) of metallic / magnetic nanoparticles

The nanoparticle particles were treated with 3-aminopropyltriethoxysilane (APTES), to anchor the amino group to its surface. For this, 90 mL of methanol and 1 mL of APTES were added to the solution of nanoparticles synthesized according to Examples 1 to 3. The mixture was stirred at 200 rpm at 50 ° C for 12 hours to generate the nanoparticle-APTES junction. Finally, it was rinsed twice with deionized water. EXAMPLE 6. Union of metallic / magnetic nanoparticles with antibacterial proteins.

2 mL of glutaraldehyde (25%, v / v) were added to a nanoparticle-APTES solution obtained according to Example 5 to make the bridge between the functionalized nanoparticuals and the protein. It was incubated at 37 ° C for 4 hours to form nanoparticle-APTES-glutaraldehyde, followed by 8 washes with deionized water.

The obtained nanoparticle-APTES-glutaraldehyde set was resuspended in 50 mL of a 5 mM phosphate buffer solution at pH 7.0. To 8.0 mL of this mixture was added 100 of an antibacterial protein or peptide solution (2.0 mg m / L) isolated from the phage, and stirred at 200 rpm, at 4 ° C for 15 hours.

EXAMPLE 7. Preparation of nanoparticles in a glass slide A coating was prepared on a glass surface (sheet) previously modified with aqua regia (HC1: HN0 ₃ ) in a ratio of 3: 1 by volume. The glass sheet was rinsed with ultra-pure water five times and three times more with ethanol by sonication. The sheet was dried in an oven at 70 ° C for 2 hours. Once the surface of the sheet was cleaned, it was immersed vertically in a solution 1% APTES / ethanol anhydride for 2 hours at 70 ° C, then rinsed again to remove excess silane and allowed to dry at 100 ° C for 2 hours plus.

Finally, the sheet was placed in a suspension of metal / magnetic nanoparticles obtained according to Examples 1 to 3, thus generating a monolayer of nanoparticles on the glass surface.

EXAMPLE 8. Preparation of an antibacterial coating The synthesis of the antibacterial coating was carried out from a suspension of metallic and magnetic nanoparticles obtained according to Examples 1 to 7. The nanoparticles were mixed with a solution at a pH between 6.5 and 7.5 of bacteriophages in concentrations between 1X10 ⁷ and 1X10 ¹⁴ UFP / mL plate forming units, to generate the coating.

The dispersion solution obtained was left in incubation for 24 hours. Finally, excess bacteriophage was removed by centrifugation. In the same way, from sheets of glass with metallic and / or magnetic nanoparticles obtained according to Example 7, sheets with the coating of the invention were obtained.

EXAMPLE 9. Antibacterial activity assay of the coating A coating of functionalized Ag nanoparticles fused with E. coli phage, obtained according to Example 8, was subjected to a bacterial growth inhibition test using the double agar technique. (Kropinski 2009. Bacteriophages: Methods and Protocols, Volume 1: Isolation, Characterization, and Interactions Editors: Clokie, Martha RJ, Kropinski, Andrew The appearance of plaques indicates the bacterial lysis due to the coating (FIG 3).

EXAMPLE 10. Antibacterial activity test of a coated sheet

A coated sheet obtained according to Example 7 was suspended in bacterial cultures of E. coli for 6, 12 and 24 hours and then were washed with PBS phosphate buffer. A Gram stain was made directly on the slide, it was observed under the microscope with the objective lOOx with immersion oil.

The number of adherent cells was determined by optical field in an area of 0.5 square cm. This was compared to the uncoated control sheet. The procedure was performed in triplicate and no adherence of the bacteria was observed in the lamina, demonstrating the antibacterial activity.

Claims

1. A coating comprising metallic and / or magnetic nanoparticles functionalized and linked with bacteriophages, with their proteins or with their peptides with antibacterial action. 2. The coating according to claim 1, wherein the metallic and / or magnetic nanoparticles are functionalized with amino, hydroxyl, carboxyl and thiol groups. 3. The coating according to claim 1, wherein the metal nanoparticles have a size between 1 and 100 nanometers. 4. The coating according to claim 1, wherein the metal nanoparticles are silver, gold, titanium, copper, iron, aluminum, platinum or tungsten. 5. The coating according to claim 1, wherein the magnetic nanoparticles are magnetite, maghemite, nickel and cobalt. 6. The coating according to claim 1, wherein the bacteriophages, their proteins and antibacterial derivatives peptides are selected from Staphylococcus sp., Steptococcus sp., Pseudomonas sp, Kelbsiella sp., Salmonella sp. Eschericia coli. 7. The coating according to Claim 1, wherein the bacteriophages are in a concentration between 1X10 ¹⁰ and 1X10 ¹² PFU / mL. 8. The coating according to claim 1, wherein the antibacterial proteins derived from bacteriophages are selected from endolysins and depolymerases. 9. The coating according to Claim 1, wherein the antibacterial peptides derived from the bacteriophages correspond to CHAP domains of endolysins. 10. The coating according to claim 1, for inhibiting the propagation or colonization of bacterial strains producing biofilms.