Patent Application
20240001003
This patent application claims priority from Italian patent application no. 102020000030230 filed on Dec. 9, 2020, the entire disclosure of which is incorporated herein by reference.
The present invention relates to a method for the antibacterial treatment of a solid surface.
With the development of the modern biomedical sector, bacterial infections of implanted medical devices have become a serious and ongoing problem. The number of infections associated with the implants of all implantable devices are around one million a year, representing a significant economic burden for society. To reduce the growing number of infections associated with implants and hospitalization costs, it is fundamental to develop materials able to prevent bacterial attachment, settlement and proliferation in these medical devices.
Among the various types of bacteria, Staphylococcus aureus and Escherichia coli are common bacteria which foul the surface of medical devices by non-specific and specific adhesion. Adhesion of the bacterial cells on the surface causes the formation of biofilm. The biofilm considerably increases survival of the bacteria and tolerance to antibiotics. Even removal of the infected implants may not fully solve the problem, because the residual bacteria can cause recurrent infections.
Three main strategies have been developed to combat the infections associated with implants: repulsion of the bacteria or a bactericidal mechanism, or a combination thereof.
The bacteria repulsion approach makes the biomaterials resistant to bacterial attack through the construction of slippery surfaces and hydrophilic antivegetative coatings.
These techniques can prevent adhesion of the bacteria for a certain period of time without killing the bacteria which, in the end, cause fouling of the surface.
The bactericidal approach instead allows the formation of surfaces able to kill the bacteria with high effectiveness in a short time by introducing a wide range of bactericidal agents.
Some of these methods use antibiotics, silver ions or ammonium ions in the medical device. However, these methods also have some limitations such as drug resistance, and result in an increase in the biofilm.
Recently, many efforts have been made to integrate the strength of the above two antibacterial approaches in one single platform and the need is felt in the art for new methods for the antibacterial treatment of a surface, in particular, for medical devices and medical supplies.
The object of the present invention is therefore to provide a new method for the antibacterial treatment of a surface which is free from the drawbacks of the known art.
This object is reached by means of a method according to claim 1 and a method according to claim 8.
The methods of the invention, advantageously, in addition to having antibacterial and/or bacteriostatic properties, have proved to be non-cytotoxic. They have also shown osteoinductive capacity, namely they are able to increase cell proliferation.
For a better understanding, the present invention is now described with reference to the attached drawings, in which:
In particular, according to a first aspect of the invention, a method is provided for the antibacterial treatment of a solid surface comprising the steps of:
The surface activation step allows both removal of the organic impurities and oxidization of the surface to be functionalized with consequent increase in density of terminal hydroxyl groups (—OH) necessary for formation of the covalent bond between the surface and the silane or the siloxane deposited in the following step.
The deposition of a silane or a siloxane can be carried out by means of vapor phase deposition, liquid phase deposition or plasma deposition and allows the formation of a single layer through covalent bonds between the surface —OH groups and the silicon of the silanes or siloxanes. This deposition makes the surface highly hydrophobic with contact angle values from 80° to 140°, preventing bacterial adhesion.
The silane or the siloxane can be selected from the group consisting of hexamethyldisiloxane, trimethyl(trifluoromethyl)silane, trimethoxy(3,3,3-trifluoropropyl)silane, trichloro(1H,1H,2H,2H-perfluorooctyl)silane, 1,1,1,3,3,3-esakis(3-fluorobenzyl)disiloxane.
In one embodiment, the method according to the present invention further comprises, prior to the surface activation step, a nanostructuring step with gamma-Fe2O3 nanoparticles. Preferably the gamma-Fe2O3 nanoparticles have a dimension of 2-2000 nm.
Advantageously, nanostructuring with gamma-Fe2O3 nanoparticles promotes osteoinduction, thanks to the magnetic field generated by the nanoparticles.
The nanostructuring step can be carried out with an iron-based reagent selected from the group consisting of iron complexes with ligands belonging to the entire class of the beta-diketones such as, for example: acetylacetonate, perfluoroacetylacetonate, trifluoroacetylacetone, benzoylacetone, hexafluoroacetylacetonate, 2-furyltrifluoroacetone, 4-methyl-2-4-heptanedione, dinaphthoylmethane, 1,3-di(2-thienyl)1-3-propanedione.
According to a further aspect of the present invention, a method for the antibacterial treatment of a solid surface comprising the step of functionalizing said surface with gamma-Fe2O3 nanoparticles is also provided. Preferably this functionalization/deposition takes place by means of a photochemical method. The functionalization can take place in liquid phase or create powders photochemically and then deposit them by plasma.
The coatings obtained with the methods of the present invention furthermore have non-cytotoxic and osteoproliferative properties.
The methods of the invention allow the treatment of a surface made of a material selected from the group consisting of titanium and alloys thereof, cobalt-chrome and alloys thereof, tantalum, steel, hydroxyapatite, plastic material, natural or synthetic fiber fabric, non-woven fabric, PEEK and polyethylene.
Further characteristics of the present invention will become clear from the following description of some merely illustrative and non-limiting examples.
Preparation of a Titanium Substrate with Hydrophobic Coating
A titanium-based sample (Ti-6Al-4V) with dimension 1×1 cm was subjected to the treatment according to the invention using trimethoxy(3,3,3-trifluoropropyl)silane as a silane reagent.
The hydrophobic coating is obtained by the formation of a monolayer having nonpolar hydrophobic terminal groups. The preparation takes place through a two-step process:
The effective functionalization of the surface was shown through contact angle measurements (table 1) and X-ray photoelectronic spectroscopy measurements (XPS) (table 2).
The XPS measurements were conducted by using a Kratos AXIS-HS instrument with Mg Kα1,2 1253.6 eV (working conditions 10 mA and 15 keV) as source. The quantitative analysis was obtained using experimental atomic sensitivity factors. Acquisition of the spectra, the processing and calculation of the quantitative chemical composition were carried out using specific software.
The SEM analysis furthermore indicates, after the silanization treatment in vapor phase, a homogeneous surface without non-uniformities or surface aggregates.
Preparation of a Titanium Substrate Treated by Nanostructuring with Photodeposition of γ-Fe2O3
The Ti samples are positioned in a quartz photoreactor, so that the surface to be treated is exposed in the direction of the light source.
A quantity of 0.1 g of Fe(acac)3 (iron tris(acetylacetonate)) is added to 500 ml of ethyl alcohol and after complete solubilization is introduced into the photoreactor.
10 ml of photosensitizer (acetone) are added and irradiated for 1.5 hours. The solution changes from red to deep yellow.
At the end of the irradiation, maintaining a constant nitrogen flow, 17 ml of NaOH 1 M are added and the solution turns darker.
The substrates are then washed with ethyl alcohol. The procedure entails at least three repetitions of the process. Lastly, the surface is dried under nitrogen flow.
Preparation of a Titanium Substrate Functionalized with Photodeposited Nanoparticles of γ-Fe2O3 and Hydrophobic
The sample obtained in example 2 was subjected to the chemical treatment as described in example 1, using trimethoxy(3,3,3-trifluoropropyl)silane as silane reagent, obtaining a surface with contact angles of approximately 100°.
The chemical-physical characterization was carried out with methods such as XPS, EDX and TEM.
The diffraction analysis of the TEM confirms by the presence of the following d-spacing values, a γ-Fe2O3 structure (2.95 Å (28%), 2.54 Å(100%), 2.09 Å (20%), 1.73 Å (10%), 1.64 Å (25%) and 1.47 Å (42%)).
The XPS analysis shows the presence of the following diagnostic peaks: C1s-F3 (293.0 eV) and C-F (689.6 eV), in addition to those typical of titanium and iron.
Antibacterial Activity of a Surface Treated with the Method According to the Invention
Antibacterial Activity of the Sample of Example 1
Method
Different procedures for sterilization of the samples were analyzed (Ethanol 70%, UV and flowing vapor sterilization in autoclave at 121°) and two different procedures for evaluation of the antimicrobial activity by means of plate count techniques (method 1-ISO 4833-2:2013. Microbiology of the Food Chain-Horizontal Method for the Enumeration of Microorganisms-Part 2: Colony Count at 30 Degrees C. by the Surface Plating Technique) and live/dead microscope acquisition (method 2) of S. aureus ATCC 29213, E. coli ATCC19138 and P. aeruginosa ATCC27853.
In all the tests the non-treated titanium sample was used as a negative control.
Preparation of the Inoculum
Preparation of the sample and evaluation of the antibacterial activity.
A sample prepared according to example 1 was inoculated with the bacteria prepared according to what was described above. The evaluation of the bacterial activity is carried out as illustrated in Acta Biomateralia 8, 2012, 904-915. doi: 10.1016/j.actbio.2011.09.031.
For each culture:
Tables 3-5 below illustrate the plate bacterial count, namely the antimicrobial activity vis-{grave over (α)}-vis S. aureus (table 3), E. coli (table 4) and P. aeruginosa (table 5).
S. Aureus sample
0
E. coli sample
Antibacterial Activity of the Sample of Example 2
Using the method described above, the antibacterial activity vis-à-vis the S. aureus of the sample obtained in example 2 was evaluated. The results are shown in table 6.
S. Aureus sample
0
E. coli sample
0
0
Osteo-Proliferative Activity
Osteo-Proliferative Activity of the Sample of Example 1
In order to evaluate the biocompatibility and proliferative capacity of the material analyzed we cultivated 1*106 mesenchymal stem cells derived from the adipose tissue (hADSCs) on the surface of a sample of example 1 for 7 days and carried out the Trypan blue count and DAPI stain at 24 h and 7 days, to evaluate the number of cells present on each material.
For the DAPI stain, the hADSC cultivated on Ti scaffolds after 24 hours and 7 days of culture were fixed in 4% PFA, washed in PBS 1X and permeabilized in TritonX-100 at 0.3% for minutes. Subsequently, the nuclei were dyed with 4′, 6-diamidino-2-phenylindole (DAPI) (1: 5000) in PBS 1X for 5 minutes and washed three times in PBS 1X. The digital images were acquired using a 20× magnification fluorescence microscope (Leica DMI4000B) in a random manner and 30 images (fields) were analyzed for each scaffold. The cell count from each image acquired was carried out using the Fiji image J software.
The results are shown in
Osteo-Proliferative Activity of the Sample of Example 2 Following the above method, the osteoproliferative activity of a treated sample as shown in example 2 was evaluated. The results are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 102020000030230 | Dec 2020 | IT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2021/061504 | 12/9/2021 | WO |
