EP2741792A1

EP2741792A1 - Process for the production by plasma of nanometric thickness coatings allowing controlled release of silver ions of other elements, or of molecules of biomedical interest, from solid products, and products thus coated

Info

Publication number: EP2741792A1
Application number: EP12773393.9A
Authority: EP
Inventors: Riccardo D'agostino; Pietro Favia; Francesco Fracassi; Eloisa SARDELLA; Ciro Costagliola; Annarosa MANGONE
Original assignee: Mediplasma Srl Dipartimento di Scienze per La Salute Dell'Universta' del Molise (UNIMOL); Universita degli Studi di Bari Aldo Moro
Current assignee: Mediplasma Srl Dipartimento di Scienze per La Salute Dell'Universta' del Molise (UNIMOL); Universita degli Studi di Bari Aldo Moro
Priority date: 2011-08-11
Filing date: 2012-08-10
Publication date: 2014-06-18
Also published as: WO2013021409A8; ITRM20110438A1; WO2013021409A1

Abstract

Process for the production by plasmochemical deposition of a film having a nanometric thickness, optionally multilayered, permitting carrying out in a controlled, uniform and long lasting way, release of substances of interest in a surrounding medium containing liquids, from a substrate including the substance to be released as micro/nano particles, or from a layer deposited on the substrate including the substance to be released as micro/nano particles, or from a layer of the substance to be released deposited on the substrate, or from a substrate that is the substance to be released optionally in the form of particles. The substances to be released can be metals, compounds having anti-bacterial properties, biologically active molecules such as drugs, hormones, vegetable extracts, peptides, lipids, protides and glucides. The layer with the substance to be released, be it organic or inorganic, is obtained by plasmochemical deposition optionally having a structure similar to polyethylene oxide (PEO) or polyethylene glycol (PEG), called PEO-like polymers, constituted, in a variable percentage da ethylene oxide units (-CH2CH2O-, EO); barrier film is obtained by depositing by plasma at least one organic or inorganic layer, optionally with a PEO-like structure, wherein chemical composition, degree of crosslinking and thickness are adjustable by the plasmo chemical deposition process parameters, and allow to adjust the release of the active substance according to specific needs. The structures on which the above said films can be deposited are: medical-surgical devices, common handworks, structures known as scaffolds, and the above defined substances to be released themselves. The invention also relates to medical-surgical devices, common handworks and scaffolds coated by a substrate and barrier layer, as well as to biologically active substances coated by at least one barrier layer.

Description

Process for the production by plasma of nanometric thickness coatings allowing controlled release of silver ions or of other elements, or of molecules of biomedical interest, from solid products, and products thus coated.

The present invention relates to the controlled release of substances of interest from materials containing the same.

Subject of the present invention is the realisation of a coating of nanometric thickness, optionally multilayered, with the ability of releasing ions, drugs, antibacterial compounds or molecules, compounds or molecules of different kind of interest in medicine and biology (such as growth factors, antibiotics, contraceptives, antithrombotic drugs, hormones, etc), and surface properties of biomedical interest (such as hydrophilic or hydrophobic character, ability to discourage or promote the adsorption of bio molecules, cells and biological tissues, increase or decrease the lubricity, increase or decrease the immune response), which are adjustable, controllable and perfectly independent from one another, suitable for improving the performance of medical devices or devices of common use.

For reasons of clarity, it should be noted that in the following, with the same meaning as "coating", the terms "layer" and "film" are also adopted. In addition, it should be noted that the layer containing the substance to be released is referred to as "matrix", while the layer that controls its release is referred to as "barrier". With the generic term "reserve" it is meant all the material or layer containing the molecule or atoms subject to release. Finally, it is specified that the term "substrate" may indicate both the support on which the multilayer according to the invention is applied, and the substance to be released on which it is applied only the barrier layer. In the present invention, the term "precursor" means the supply gas of the plasmochemical deposition processes.

In U.S. 2009/0035341 the barrier layer only has the property of control of permeability to oxygen. In WO 2010/084315, instead, it is not present any barrier layer external to the substrate loaded with antibacterial substances. A scheme of the possible configurations of the multilayer according to the invention is shown in Figure 1 , which reports embodiments where the substance to be released is in the form of free particles, or included in the substrate or dispersed in a coating deposited on it, or in the form of coating deposited on the substrate, or constitutes the substrate.

Silver, as an example of the substance to be released, has broad spectrum of activity against microorganisms; in the case of multilayer coatings containing silver, the antibacterial properties are derived from the release of Ag⁺ ions from the layer adherent to the substrate, release that can be dosed.

Other metals or oxides also have antibacterial properties similar to silver, such as for example copper, zinc, titanium dioxide (TiO₂)_, titanium, gold, carbon, gallium, as well as organic substances of various kinds, including antibiotics and the bacteriostatics.

The surface properties of biomedical interest (eg stimulation or inhibition of cell adhesion) of the multilayer relate to the surface of the outer layer of the coating, and can be predetermined, for example to promote or discourage the adsorption of organic molecules and/or the adhesion of cells and tissues. In the first case it is possible to speak of coating cell and/or protein-adhesive, in the second case of films unfouling.

To the outer layer it is also given the ability to control the amount of released silver or of other substances included in the multilayer.

All the outer layers capable of modulating the release, subject of the present invention, are made by means of "cold" plasmas; the substrate and the inner layers of the multilayer can be realised by plasma, or in some other way, depending on the application.

The plasma is partially ionized gas, rich in active species (eg, atoms, ions, electrons, radicals) generated by fragmentation, ionization and recombination of individual molecules, atoms, ions of the starting gas, through the application of energy (eg heat, or electromagnetic fields). The state of plasma, present in nature (eg stars, fire, lightnings, aurora borealis), can be artificially produced in conditions of equilibrium (eg plasma welding, torches for the incineration of waste) or of non-equilibrium thermodynamics (eg fluorescent lamps, plasma TV, etc.). In the first case it is possible to speak of "hot" plasmas, characterised by high temperature, even about 10.000 K. The non-equilibrium plasmas, or "cold" plasmas, of interest to the present invention, have instead temperature close to ambient temperature, and are of interest for the treatment of materials in a variety of applications, including those in the biomedical field.

In suitably configured reactors it is possible to expose the materials to cold plasmas, by modifying the surface by processes of: etching (chemical ablation of the material); thin film deposition (PE-CVD, Plasma Enhanced- Chemical Vapour Deposition); treatment (grafting of chemical groups to the surface of the substrate); sputtering (physical ablation of the material). By cold plasma it is possible to treat in a very versatile way thermolabile conventional materials, such as polymers, paper, leather and textiles. By varying the experimental parameters (eg. nature and flow of the supply gas/steam, pressure, power, etc.) it is possible to vary the chemical/physical surface properties of materials (eg hydrophobic/hydrophilic character, biocompatibility, etc.), leaving unchanged structure and massive properties. This is a real advantage for materials of biomedical interest (eg prosthesis, biosensors, devices, etc,) for which the interactions with the host organism or biological fluids is mainly localized at the interface material/biological environment, also being able to modulate the antigenicity.

The plasmo chemical processes allow to modify at room temperature the chemical features and the properties of the most superficial layers of the materials, leaving unaltered structure and massive composition. Other advantages are: the versatility, the absence of solvents, the minimum amount of reagents, the negligible environmental impact, the low cost of the process, the ability to scale the processes to products of large dimensions, and the integrability in production lines.

Coating deposited in plasmas supplied with volatile monomers characterized by ethylene oxide units (-CH₂CH₂O-, EO), may exhibit similar chemical structure to the polymers polyethylene oxide (PEO) or polyethylene glycol (PEG), and for this reason are called PEO-like polymers. The concept of "PEO-like" film is extended in this invention to films that have a percentage of ether groups, measurable with the X-rays Photoelectron Spectroscopy (XPS) technique of between 10% and 100%, then even very distant for chemical composition from PEO and PEG polymers. Because of their great affinity for water PEO-like films with a percentage of ether groups between 65% and 100% with respect to all the other features of the carbon (carboxyl, carbonyl, alcohol, etc.) behave as hydrogels and discourage, in vitro, the adsorption of proteins, cell adhesion and biofilm formation (unfouling effect).

Supplying the plasma with gas/vapors of other substances, it is possible to deposit coatings of different nature, which are also used to modulate the release of substances from substrates of biomedical interest and, simultaneously, to modulate the surface properties.

By plasma it is also possible to deposit nano composite coatings, ie in which particles (clusters) of silver, of other metals, of oxides of the same or of other compounds (eg. hydroxyapatite) nanometers in size are dispersed in matrices not necessarily of PEO-like nature but also of other organic and inorganic nature. Coatings containing clusters of silver dispersed in PEO-like matrix, called Ag/PEO-like, in an aqueous ambient release Ag⁺ ion, with proven antibacterial effect.

In addition to the aforementioned antibacterial activity, it is also possible to deposit, for example on medical devices or on structures known as scaffolds, coatings in which hormones are dispersed, and drugs, proteins, and anything else that may be able to interfere in biological processes, controlling the release and thereby modulating the effects.

The present invention makes it possible to modulate the released amount, of silver ions or of other ions or of other substances, and the consequent effects of biomedical interest. For example, the blocking of receptors for various growth factors (epithelial, vascular, nervous) or, on the contrary, the stimulation of said receptors. Still, it is possible to modulate the immune response of prosthetic plants (hip prosthesis, cardiac valves, etc..) and medical devices not to stay (cat ethers, vascular accesses, etc.).. The field of application is, therefore, infinite, or at least as broad as it is the knowledge about the possible interactions with living matter.

In the case of ions or other antibacterial substances, the barrier layer allows to benefit from the properties of the substances, but to reduce the possible cytotoxic effect of some of them, for example of silver ions, against eukaryotic cells, thanks to the nano molar and lower concentrations used. Furthermore, since the barrier film is deposited by plasma, its chemical-physical characteristics can be varied in a targeted and simple manner by changing the deposition parameters and, therefore, giving the film itself varaible barrier and biological properties, peculiar of the process used and also totally different from those of conventional materials in commerce.

In the case of Ag/PEO-like and PEO-like coatings, these have been deposited by plasma on various substrates, including devices of biomedical interest, in a radiofrequency (RF, 13.56 MHz) plasmo chemical reactor. In order to deposit Ag/PEO-like films in this reactor working conditions must ensure, at the same time, the sputtering of atoms and clusters of silver from the target electrode of the reactor, and the deposition on the other electrode, of a PEO-like coating that incorporates the silver clusters. The configuration with two electrodes may be replaced by one with three or more electrodes, depending on the needs of the process or application. In optimized conditions, when power increases and when the working pressure and the flow of precursor decrease, the degree of crosslinking of the PEO-like matrix, the incorporated cluster size and the total amount of incorporated silver increase. When the deposition time increases the thickness of the coating also increases.

The silver content of the coating can be measured, for example as atomic percentage on the elements Ag, C and O, with the XPS technique. By varying the material of the target electrode and the precursor, then the nature of the clusters and of the organic matrix, it is possible to deposit other types of nano composite coatings, for example containing clusters of other metals such as copper, zinc, titanium, gold, gallium, carbon, or oxides, such as titanium oxides, or ceramic materials such as hydroxyapatite. The Ag/PEO-like coating constitutes the reserve of silver at the surface of the coated device; the thickness of the coating can vary from 10 nm to several microns (up to 50 microns), depending on requirements. The release of Ag⁺ ions takes place in an aqueous environment and is a function, inter alia, of the quantity of silver incorporated in the coating and the chemical nature of the matrix in which clusters are incorporated. Measurements of release of silver in cell culture media by Ag/PEO-like coatings were conducted by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), as shown in Figure 2. The measurements showed that the release rate in aqueous media gradually decreases over time, and varies depending on the chemical nature of the matrix of the coating, the amount of incorporated silver, and the thickness.

Suitably selecting the parameters of deposition of the Ag/PEO-like coating it is possible to control: the quantity of incorporated silver; the degree of crosslinking of the PEO-like organic phase in which silver is finely dispersed; and the silver cluster size. Therefore, depending on the characteristics of the deposited film it is possible to modulate the antibacterial effect by acting on the quantity of incorporated silver and offering, in this way, various technological solutions depending on the area of application of the device so coated.

The mechanism of release of silver ions follows the kinetics of a typical drug release system of the matrix type. It is supposed occurring first the diffusion of silver through the PEO-like matrix soaked with water, up to reach first the material/biological environment interface, and then the aqueous medium. This mechanism is affected by the degree of crosslinking of the matrix, the amount of silver in the coating, and the cluster size. Film containing very high amounts of silver, for example, may release silver ions in a non-controlled manner.

The coating that contains silver, or other bioactive substances, may also comprise a matrix different from the PEO-like one described above, for example of the siliconic type, silicon oxide (SiOx), fluorocarbon (CFx), hydrocarbon (CHx), acrylic (COxHy), or of different type, not necessarily produced by plasma. Ions and substances that act against bacteria may also have slightly cytotoxic effect, especially if with locally high concentration, higher than those (minimum inhibitory concentration) that guarantees the antibacterial effect (0.5-10 mg/l in the case of Ag⁺ ion).

The present invention, therefore, concerns the application of a coating with barrier effect, for example of PEO-like type, having controlled thickness (5- 10000 nm) and chemical composition, above the Ag/PEO-like coatings, or in any case, above the coating or the substrate containing the bioactive substance/substances to be released. In the specific case of multilayer films with PEO-like type barrier on Ag/PEO-like films, by varying with the deposition parameters the density of EO units (then the crosslinking of the PEO-like matrix, measurable with XPS) in PEO-like coatings, it is possible to modulate the release of Ag⁺ ions, or other substances; further it is possible to modulate also the adsorption of proteins and the adhesion of eukaryotic cells on the coating. A highly crosslinked PEO-like coating (low density of EO units) will significantly decrease the amount of silver ions released and promote protein adsorption, adhesion of cells and tissues on the coated device; if slightly cross-linked (high density of EO units), instead, the coating will discourage the adsorption of organic molecules and the adhesion of cells funfouling effect).

The deposition of the PEO-like coating can be made in the same reactor, immediately after the deposition of the Ag/PEO-like coating, under conditions that prevent the sputtering of silver, for example: by replacing the electrode of silver with an inert one (for example of steel), or increasing the working pressure, or by shielding the target electrode with a screen (shutter). With decreasing power, increasing the flow of precursor in the supplied mixture, and increasing the pressure, the amount of EO units in PEO-like coating increases and then the unfouling character of the same increases; with increasing the deposition time the thickness of the coating increases.

One of the most important advantages of the barrier coatings forming a subject of the present invention is that, for example in the case of nano composite coatings containing silver, they allow to adjust the amount of silver released in situ by the nano composite coating, in such a way as to be still effective against bacteria, but not cytotoxic for eukaryotic cells. In order to obtain other surface properties, the barrier coating deposited by plasma may be for example of the siliconic type, silicon oxide (SiOx), fluorocarbon (CFx), hydrocarbon (CHx), acrylic (COxHy), or of different type.

The antibacterial activity of the Ag/PEO-like films was tested on lines of Pseudomonas aeruginosa and Staphylococcus Epidermidis bacteria. In the first case, using a test of bacterial adhesion (4 * 107 cells/ml, with stirring), it was observed that films containing 3.5% of silver knock down of 100% the adhesion of bacteria after 2 hours of incubation compared to untreated substrates, metals and polymers and ceramics, of various types. In experiments conducted with the S. Epidermidis ATCC 35 984 species (3x108 CFU/ml) both in static and dynamic conditions (room under flow with flat and parallel plates), after 2 hours of incubation it was observed that films containing 24.4% of silver knock down 80% of bacteria adhering to the surface of the substrates.

The present invention allows, instead, to realize a multi-layer coating with controlled release of silver ions, uniform and durable in a humid environment, exploiting the barrier film. The multilayer film is realised entirely by plasma with a multi-stage process. The invention also allows to attenuate the release over time so as to reduce or eliminate the cytotoxic effect of silver, while retaining the antibacterial.

The invention is based on the deposition by plasma, as one method of synthesis, of the barrier film: the process ensures, therefore, high versatility of application both because it is independent of the nature of the coated material, and because the antibacterial properties (or in general the release properties), biocompatible and unfouling of the multilayer can be varied independently. In fact it is subject of the present invention a process for the realisation on tools or devices of bio-medical nature (such as prosthesis, catethers, intraocular lenses, filters for hemodialysis, cardiac valves, syringes and other medical devices) and on common handworks (such as filters for air conditioning systems, toothbrushes, long-lasting razor blades, fluid circulation tubes), by plasmochemical deposition, of an organic or inorganic film optionally with PEO-like structure and having antibacterial properties, or with the possibility of release for medical devices also of drugs or compounds having another type of action (therapeutic, hormone, etc.) characterized by the fact that the deposited film is multi- layered and is obtained substantially by the following operations:

- deposition by sputtering of a target of silver (or other metals or bioactive oxides or hydroxyapatite) and by PE-CVD at the same time, of a first layer of inorganic or organic, optionally PEO-like matrix, in which clusters of the metal are embedded;

- deposition by PE-CVD of at least a second organic or inorganic layer, optionally a PEO-like layer, with chemical or thickness characteristics suitable to contain and control the release of silver ions or of different kind of ions (barrier film).

The resulting multi-layer will then be able to control and make uniform and long lasting the release of antibacterial ions Ag⁺ (or of other ions or compounds with antibacterial effect, or of any other substance for which it is required to reach a concentration which is effective to exert an effect of therapeutic type) in liquid or wet environments due to the chemical nature and thickness of the barrier layer (crosslinked and/or thick films reduce the release).

The amount of silver released also depends on the total quantity of silver incorporated in the nano composite film and on its thickness.

The plasmo-chemical depositions reported for the present invention were performed with reactors powered with an alternating electric field with frequency of 13,56 MHz In this case, the first layer can be obtained by exposure to the process of sputtering/PE-CVD for a time period sufficient to realise a nano composite coating thickness comprised between 5 nm and a few microns. Similar experiments can be carried out by powering the plasma with sources at lower frequencies (tens-hundreds of kHz) and higher than 13,56 MHz, always obtaining coatings with comparable performance.

The layer or layers subsequent to that containing the substance to be released can be obtained by plasma exposition for a time period sufficient to realise an appropriate thickness, under experimental conditions such to control chemical nature and degree of crosslinking of the film. It is also possible to create coatings with a chemical composition gradient (ie with a higher crosslinking at the beginning of the process, and a lower crosslinking at the end), or more layers of chemical natures completely different between them, multifunctional (eg antibacterial/unfouling; antibacterial/cell-adhesive; antibacterial/hydrophobic, antibacterial/hydro- philic, etc.).

More generally, the present invention relates to a process for the production, by plasmochemical deposition of a barrier film, optionally multilayered, optionally with a PEO-like structure that allows for a controlled, uniform and sustained release of preset substances.

The layer with the substance to be released is obtained by depositing of an inorganic or organic layer, optionally with a PEO-like structure. The substances to be released, coated by the barrier film, can be included: in a substrate in the form of micro/nano particles; or in a layer deposited on the substrate, in the form of micro/nano particles; or in the form of coating deposited on the substrate; or as an integral part of the same substrate, or as distinct micro/nano particles.

These substances may include metals and compounds having antibacterial properties and biologically active molecules, such as drugs, hormones, vegetable extracts, oligo peptides, lipids, protides and glucides. The structures on which the above said deposits can be made are represented by medical-surgical devices, common handworks, structures known as scaffold, and the substances to be released themselves.

The present invention is not limited to the process described above but extends, in addition to combinations of plasmo chemical processes different from those mentioned so far, also to the coated products resulting from the execution of the process. Therefore the subject of the invention are also medical-surgical devices, common handworks and scaffolds coated by the layer containing the substance to be released and at least one barrier layer, as well as the biologically active substances with at least one barrier layer deposited by plasma.

In the process according to the invention, the gas or vapor precursors for deposition by plasma of the barrier film, are: organic compounds that do not contain oxygen of the classes of alkanes, alkenes and alkynes, or compounds containing oxygen not polymerisable by plasma such as O2_, CO2 and H₂0, alone or mixed with polymerisable precursors, or compounds containing oxygenated groups for example with structure CH₃- (OCH₂CH2)n-OCH3, ethers, crown ethers, other compounds that contains oxygenated groups such as glycols, further ethers, alcohols, carboxylic acids, aldheydes, anhydrides, ketones, phenols; or compounds containing nitrogen not polymerisable by plasma, such as N2_, NH₃, to be used alone or mixed with other precursors that are polymerisable by plasma or nitrogenous compounds such as primary, secondary and tertiary ammines, allyl amines, diamminocyclohexane, N-isopropylacrilammide, or compounds of inorganic nature such as silicon-containing organic molecules, or fluorine compounds of carbon.

Figure 1 schematically shows possible embodiments of the multilayer according to the invention which are:

A) The substrate includes the substance to be released through the barrier coating as micro/nano particles;

B) The substance to be released through the barrier coating is included as micro/nano particles in a coating deposited on the substrate;

C) The substance to be released through the coating barrier is in the form of a coating deposited on the substrate;

D) The substrate constitutes the substance to be released through the barrier coating;

E) The barrier coating is deposited directly on particles of the substance to be released.

Figure 2 shows ICP-MS measurements of release of silver ions by Ag/PEO-like coating with comparable crosslinking (EO% between 20 and 40%) and content of silver (XPS) equal to 1%, 3% and 5% . The coating containing more silver release larger amounts of silver ions over time.

Figure 3 shows the bactericidal effect ("zone of inhibition test ") towards Staphylococcus epidermidis RP62A ATCC 35 984 (slime producer, Gram+), of substrates of PET film coated with Ag/PEO-like PEO40%Ag†%, ie with the PEO-like matrix consisting of 40% of EO units, and containing 1 % silver.

Some substrates are coated by PEO-like coating barriers with different composition (70%, 40%, 20% EO) and thickness (60 nm and 15 nm), which modulate the antibacterial effect.

Figure 4 shows ICP-MS measurements of release of silver ions by Ag/PEO-like coating with 5% of silver, coated by two PEO-like barrier coating with different crosslinking (EO 40% and 20%) and different thickness (15 nm and 60 nm). When increasing the thickness and the crosslinking of the coating barrier the release decreases.

As has already been said, Figure 3 shows the bactericidal effect, measured with the zone of inhibition test, of substrates of polyethylene terephthalate (PET) coated with Ag/PEO-like film PEO40%Ag1 %, ie with the PEO-like matrix composed of 40% of EO units, and containing a low amount of silver, 1 %, in order to demonstrate the efficacy even at a low rate of release. Some substrates are coated by PEO-like barrier coatings with different composition (70%, 40%, 20% EO) and thickness (60 nm and 15 nm), that modulate the antibacterial effect.

The data of Figure 3 show that the substrate as such (TQ PET) and the PEO40% barrier coating obviously do not have antibacterial effect. The PEO40% Ag1 % coating, 80nm thick, without barrier coating, shows the maximum antibacterial effect, measured by a zone of inhibition of more than 5 mm after 72 h of incubation at 37 °C. The test was continued for one week, to verify possible late effects. When the PEO40% Ag1 % film is covered by barrier coating the zone of inhibition is reduced, to witness the reduced release of silver ions, when the crosslinking of the PEO matrix is increased (the lower the PEO% value, the higher the crosslinking) of the barrier film and its thickness. The data ICP-MS in Figure 4 confirm how the release of Ag⁺ ions by Ag/PEO-like coatings is modulated as a function of crosslinking and thickness of the PEO-like barrier layer. EXAMPLE 1

The reactor used for the depositions of the present invention is substantially constituted by a single reaction chamber in stainless steel, connected to the supplying and control equipment such as:

- means to realize vacuum;

- system pressure reading within the reaction chamber (eg. Baratron);

- generator of radio frequency (RF, 13.56 MHz);

- an impedance adapter (matching network);

- supply systems to the reaction chamber (which include the reserves of the precursor) of reagents in the gaseous or vapor state;

- a flat silver electrode, with earthed shield, which is replaceable with an electrode of stainless steel (or that is darkened with a shutter), connected to the radio frequency generator, disposed within the reaction chamber, which ensures also the sputtering of silver, of smaller dimensions than the one connected to earth ('ground electrode);

- an electrode connected to earth (ground), flat, inside the reaction chamber, on which the medical-surgical devices or the common handworks to be treated are arranged.

This reactor could also be provided with two or more target electrodes for sputtering at the same time other metals or oxides or bioactive ceramic materials on the surface of the material to be treated. All target electrodes in this case should be replaceable with inert electrodes such as those of steel or totally shieldable with the use of shutters.

The use of such a reactor used for the films shown in the example of the present invention does not preclude the use of other plasmo chemical and not methods for the production of materials or layers that are able to act as "reserves" for the controlled release of bioactive molecules or atoms. For example it would be possible to use magnetron reactors, plasma spray systems, plasmas at atmospheric pressure.

EXAMPLE 2

The invention provides for the deposition of a coating consisting of two or more superimposed organic or inorganic layers, the first of which containing metal (eg. silver) clusters or bioactive or ceramic oxides, while the subsequent layers only have barrier effect or also other effects such as unfouling effect, increased biocompatibility, reduce antigenicity, variation of the coefficient of friction or effects that can be controlled with specific stimuli such as variations of temperature or pressure or acidity of the medium (smart materials). The process for the deposition of films reported as exemples of this invention involves the following steps:

a) preparation and introduction of biomedical devices (such as prosthesis, catethers, intraocular lenses, hemodialysis filters, parts of biosensors) or of common handworks (such as filters for air conditioning, toothbrushes, long-lasting razor blades) on special compartments arranged in different parts of the reactor (floating) or directly on the ground electrode;

b) formation of process vacuum in the reaction chamber (<10³ Torr);

c) check that there is no contamination of air and/or moisture inside the reactor containing the materials;

d) setting of the process parameters in the reaction chamber including: choice of the gas or of the feed mixture; flow of the feed gas, pressure of the gas; working power; water cooling of the RF electrode; bias potential of the target electrode/electrodes;

e) simultaneous placing of gas/vapor reactants in the reactor;

f) ignition of plasma;

g) plasmo chemical deposition for a time sufficient for the formation of the first organic or inorganic coating having a thickness between 5 nm and a few microns and simultaneous formation, inside it, of nanometric clusters of silver or other metals or bioactive or ceramic oxides by sputtering;

h) turning off the plasma, after a time sufficient to achieve a film of the desired thickness, and evacuation of the reactive gases from the reaction chamber;

i) switching off of the pumping and air inlet systems;

j) opening of the reactor with replacement of the target/targets of silver (or other metals or bioactive or ceramic oxides) with those inerts (for example steel) in case a configuration that includes the use of shutters for covering the electrode (or electrodes) target is not available;

k) repeat the procedures described in paragraphs b)-c)-d) e) and f);

I) plasmo chemical deposition for a time sufficient to the formation of the second organic barrier coating, possibly also characterized by other properties (unfouling, biocompatibility, etc..) with a thickness in the range between 5 and 5000 nanometers;

m) switching off of the plasma and evacuation of reactive gases from the reaction chamber;

n) switching off of the pumping and air inlet systems;

o) extraction of the treated biomedical devices or handworks.

In case shutters are available the procedure in paragraphs i) and j) is not required and after coating the target/targets with such devices it is possible to pass with order directly from the procedure k) to o). This procedure obviously changes depending on the type of technology used for the obtainment of the "reserve" film or material.

EXAMPLE 3

According to preferred embodiments of the process, it is expected that the treatment phase of biomedical devices such as those shown as an exemple of the following invention (PEO-like and Ag/PEO-like) obtained in the reactor described in Example 1 , is carried out in the following experimental conditions:

- pressure comprised between 1 mTorr and 50 Torr;

- power comprised between 0,001 and 20,00 W/cm² (relative to the surface of the electrodes);

- flow of the gases involved in the process comprised between 0,01 and 10.000 seem (standard cubic centimeters per minute);

- treatment time between 3s and 10 hours.

Varying the supply gas of the plasma and the deposition conditions it is possible to obtain thin films with variable composition in continuous with the deposition conditions, with absolutely unique characteristics; using precursors like ethylene glycols it is possible to deposit organic coatings with unfouling characteristics, ie resistance to the adhesion and contamination with organic and biological substances in aqueous or wet ambient. Coupling a sputtering process with a conventional PE-CVD process it is possible to produce nanocomposite coating, ie constituted of (metal, ceramic, oxides or polymeric) nanometric clusters included in the thin film of organic or inorganic nature.

EXAMPLE 4

The XPS analysis of the nanocomposite films with barrier described above (PEO-like multi-layer on Ag/PEO-like) showed the presence of a quantity of silver variable between 1 and 40% (in atomic percent) and a good degree, uniform and compliant, of covering nanocomposite films using the barrier film. Measurements of wettability on films with and without barrier reveal a contact angle with water ranging between 20±3° to 95±5° depending on the amount of silver incorporated into the organic matrix and the roughness of the deposited film. Measurements of the release of silver in aqueous media were conducted using ICP-MS on samples from cell culture media. These measurements revealed that the release of silver decreases gradually over time passing, for example, for films containing 5% of silver, from 0,15ppm^*h^{" *}cm^"2 to about 0,005ppm^*h^{"1 *}cm^"2 after 6 days of exposure to the culture medium DMEM (Dulbecco's Modified Eagle's Medium). The bactericidal effect was evaluated by means of the zone of inhibition tests of materials covered with films containing silver, prepared under various experimental conditions and coated with various barrier films.

The biological test was carried out with agar medium (Tryptic Soy Agar, TSA) containing 1x10⁸ cfu/mL of Staphylococcus epidermidis RP62A ATCC 35984 (slime producer, Gram +). The test was performed after 72 h of incubation and continued for one week in order to verify possible late effects. The size of the ring that is formed around the modified material gives a measure of the effect of the antibacterial material. In Figure 3 are reported the results of the zone of inhibition test performed on films containing the least amount of incorporated silver (silver XPS 1 %). From the results shown it is clear that materials not containing silver such as PET as such (PET TQ), and PEO-like films containing 40% (PEO40%) of EO ether groups deposited on PET did not show antibacterial effect. The histogram in Figure 3 shows that the reduction of the effect by means of the antibacterial barrier film depends both on the amount of EO groups (PEO character) and on the thickness of the barrier film, shown in brackets. It follows that thicker (60 nm) and/or more crosslinked (PEO20%) barrier films cut down significantly the release of Ag⁺ ions and the antibacterial effect, almost to cancel it. This effect is confirmed by ICP- MS measurements; the silver released was quantified in cell culture medium DMEM (Dulbecco's Modified Eagle's Medium) with cell line of 3T3 fibroblasts at different incubation times, taken during an experiment evaluation of the cytotoxicity of these materials. The release was monitored after 24, 48 and 72 hours of exposure of the film to the cell culture medium, incubated with the cell line of 3T3 fibroblasts. The portions of the culture medium were digested with HNO3 concentrated hot, brought to dryness and taken up with a solution 2% v/v solution of HNO₃. During exposure to the aqueous medium, films containing the highest concentration of silver (PEO20% Ag5%) release more silver over time, compared to films that contain less (Figure 2). On the other hand, for modulating the release from the films which contain the greatest amount of silver, more crosslinked (lower percentage of ether groups, lower PEO character) and thicker barrier film will be needed (Figure 4).

The cytotoxicity experiments carried out in conjunction with the determination of the release have allowed to verify that, in the absence of barrier films, inhibition of adhesion and cell proliferation of 3T3 fibroblasts (line particularly sensitive to silver) occurs on Ag/PEO-like coatings. In the presence of barrier films, however, the 3T3 fibroblasts adhere and proliferate undisturbed at the base of the wells for cell culture, around them and on the treated materials. These results confirm that it is possible to reduce the cytotoxic effect of silver released without renouncing to its antibacterial effect.

Claims

1. Process for realizing, by plasmochemical deposition, a film optionally a multi-layer film (barrier film), having a nanometric thickness permitting carrying out in a controlled, uniform and long lasting way, release of substances of interest in a surrounding medium containing liquids, from a substrate including the substance to be released as micro/nano particles, or from a layer deposited on the substrate including the substance to be released as micro/nano particles, or from a layer of the substance to be released deposited on the substrate, or from a substrate that is the substance to be released possibly in the form of particles, wherein the substance to be released can be metals, compounds having anti-bacterial properties, biologically active molecules such as drugs, hormones, vegetable extracts, peptides, lipids, protides and glucides; the layer with the substance to be released (matrix) , being it organic or inorganic, is obtained by plasmochemical deposition optionally having a structure similar to polyethylene oxide (PEO) or polyethylene glycol (PEG), called PEO-like polymers, constituted, in a variable percentage by ethylene oxide units (-CH₂CH₂0-, EO); barrier film is obtained by depositing by plasma at least one organic or inorganic layer, optionally with a PEO-like structure, wherein chemical composition, degree of crosslinking and thickness are adjustable by the plasmo chemical deposition process parameters, and allow to adjust the release of the active substance according to specific needs; and the structures on which the above said films can be deposited are: medical-surgical devices, common handworks, structures known as scaffolds, and the above defined substances to be released themselves.

2. Process according to claim 1 wherein, on medical-surgical-devices, common handworks, structures known as scaffolds, and the same above defined substances to be released themselves, is realized by plasmochemical deposition of a multi-layer coating of organic or inorganic polymer, optionally with a PEO-like structure, substantially by the following operations: - depositing a first layer (matrix) optionally a PEO-like layer and at the same time depositing silver or another metal or bio-active oxide, having anti-bacterial^' properties, by sputtering, creating clusters of the same in the deposited substrate;

- depositing a film with one or more layers, optionally a PEO-like layer, (barrier layers), being able making controlled, uniform and long lasting releasing, in liquid or wet ambient, antibacterial ions deriving from silver (Ag⁺) or other metals or bioactive oxides, depending on the chemical nature of deposited material, and releasing an amount of silver, other metal or bioactive oxide, having anti-bacterial non cytotoxic properties.

3. Process according to claim 2, wherein deposition of the first layer (matrix) is realised by at the same time depositing silver, copper, zinc, titanium, gold, gallium, carbon, titanium oxides or ceramic materials such as hydroxyapatite, and combinations thereof.

4. Process according to any of claims from 1 to 3, wherein the barrier film, optionally multilayered, is obtained by plasma exposition for a time period sufficient to to realize on each layer a thickness comprised between 5 nanometers and 50 micrometers, under such experimental conditions to control chemical nature and degree of crosslinking of the film.

5. Process according to any of claims from 1 to 4, wherein the barrier film, optionally multilayered, is realised with a chemical composition gradient in particular with a higher crosslinking at the beginning and with a lower crosslinking at the end, or with a plurality of multifunctional layers having completely different chemical features from each other, such as antibacterial/unfouling layer, antibacterial/cell-adhesive layer, antibacterial/hydrophobic layer, and antibacterial/hydrophilic layer.

6. Process according to any of claims from 1 to 5, wherein deposition of unfouling PEO-like polymer barrier film layers is carried out by plasma under bland deposition conditions, that is power lower than 20 Watt, precursor flows higher than 1 seem, gas pressure higher than 50 mTorr, under alternate current with frequencies in the order of MHz or KHz.

7. Process according to claim 6, wherein low frequency plasma is supplied in radio frequency.

8. Process according to any of the previous claims from 1 to 7, wherein the first layer (matrix) containing the substance to be released is obtained by plasma exposition and silver sputtering for a time period sufficient to realise a layer thickness comprised between 10 nanometers and 50 micrometers.

9. Process according to any of claims from 1 to 8, wherein the barrier film, optionally multilayered, is obtained by plasma exposition under power, pressure, supply gas, variable conditions, for a time period sufficient to realise a layer thickness comprised between 5 nm and 50 micrometers with chemical and thickness features suitable to realise barrier effect.

10. Process according to any of claims from 1 to 9, wherein gas or vapor precursors for plasma deposition of the barrier film, are: organic compounds that do not contain oxygen of the classes of alkanes, alkenes and alkynes; or compounds containing oxygen not polymerisable by plasma such as 0₂, CO2 and H₂0, alone or mixed with polymerisable precursors; or compounds containing oxygenated groups for example having the structure CH3-(OCH2CH2)n-OCH3, ethers, crown ethers, other compounds containing oxygenated groups such as glycols, further ethers, alcohols, carboxylic acids, aldheydes, anhydrides, ketones, phenols; or compounds containing nitrogen, not polymerisable by plasma, such as N₂, NH₃, to be used alone or mixed with other polymerisable precursors by plasma; or nitrogenous compounds such as primary, secondary and tertiary ammines, allyl ammine, diamminocyclohexane, N- isopropylacrilammide; or compounds of inorganic nature such as silicon- containing organic molecules; or fluorinated compounds of carbon.

11. Medical-surgical devices, characterised in that they are coated by the multilayer film obtainable by the process of claims from 1 to 10.

12. Medical-surgical devices, according to claim 11 , such as syringes, prosthesis, catethers, contact lenses, cardiac valves, intraocular lenses and hemodialysis filters.

13. Common handworks, characterised in that they are coated by the multilayer film obtainable by the process of claims from 1 to 10.

14. Handworks according to claim 13, such as hemodialysis filters, filters for air conditioning systems, toothbrushes, long lasting razor blades, fluid circulation tubes, operating room sterile towels and tissues.

15. Structures known as scaffolds, characterised in that they are coated by a multilayered film that is obtainable by the process of claim 1 , in the first layer of which (matrix) molecules are dispersed that can interfere with biological processes, such as drugs, hormones, vegetable extracts, oligopeptides, lipids, protides and glucides, release of said molecules being controlled and their effect modulated.

16. Biologically active substances, optionally in the form of particles, characterised in that they are coated by one or more layers of barrier film obtainable by the process of claim 1 , release of said substances being controlled and their effect modulated.