WO2010111790A1 - Biocidal polymers, methods of preparation thereof, and methods for disinfecting and/or sterilizing objects - Google Patents

Abstract

There are provided biocidal polymers. Such polymers can be prepared by exposing at least one surface of a polymer to an ozone treatment (for example dry ozone). There are also provided methods for disinfecting and/or sterilizing at least one surface as well as methods for inactivating microorganisms.

Description

BIOCIDAL POLYMERS, METHODS OF PREPARATION THEREOF, AND METHODS FOR DISINFECTING AND/OR STERILIZING OBJECTS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to US Provisional

Application No. 61/165,589 filed on April 1 , 2009, which is hereby incorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

[0002] The present disclosure relates to the field of disinfection and sterilization of objects. In particular, the present disclosure relates to a method for disinfecting and/or sterilizing at least one surface of an object. The disclosure also relates to biocidal polymers as well as a method of preparation thereof.

BACKGROUND OF THE DISCLOSURE

[0003] Inactivation of different types of microorganisms by gaseous ozone is successfully achieved provided, as is well known, the gaseous phase is strongly humidified. Ozone is a strong oxidative compound when dissolved in water and, because of this, is known to be an efficient disinfectant for inactivating even chemically resistant microorganisms. Ozone can be an adequate solution where other disinfectants fail. Its oxidative potential is higher, for example, than that of hydrogen peroxide and hypochlorite. Ozone was recommended as an alternative to chlorine for water treatment. On practical ground, ozone is easily generated on-site from dry air or O₂ through high-voltage corona discharges at near ambient pressure and temperature. Even though ozone has been utilized in the disinfection of water for a long time, there has been a limited interest for its use in the gaseous form for disinfection and, until only very recently, for sterilization purposes (TSO₃™ sterilizer).

[0004] The growing use of heat-sensitive polymer instruments in hospitals has created new challenges in the area of sterilization. Conventional dry- and moist-heat methods, such as the Poupinel (Pasteur) oven and the autoclave respectively, can heavily damage thermosensitive materials. This is not the case with chemical sterilants such as, for example, ethylene oxide (EtO), but EtO requires long exposure and vent times (more than 1O h total) in addition to being toxic and, on the long run, carcinogenic and detrimental to the environment. As a result, EtO is already banned in many countries. Such considerations have led researchers to look for alternate efficient sterilization processes that would hopefully operate at low temperature, be able to inactivate rapidly (≤ 1 h) all kinds of microorganisms with low damage to medical devices (DMs) and be harmless for man and his environment. Under such requirements, both gaseous-plasma and ozone sterilization seem to offer substantial promises for sterilizing thermosensitive MDs.

[0005] From its inception and for a rather long period of time thereafter, the use of O₃ as a gaseous biocidal agent in ambient air was limited to concentration levels close to 1 ppm (as a matter of fact, 0.1 ppm was determined to be the occupational (40h/week) maximum safety-value as far as toxicity for man is concerned). In a study performed on sprayed bacterial suspensions in a room at known temperature and relative humidity (RH) values, Elford and Van de Eude (J. of Hygiene 42, 240-265,1942), determined that concentrations in O₃ in excess of 1 ppm in the room with 60-80% RH were better conditions, relatively to a dry atmosphere, for the inactivation of bacteria. Kowalski et al (Ozone Sci. & Eng. 20, 205-221 , 1998) studied the influence of higher concentrations of airborne ozone against E. coli and S. aureus vegetative bacteria. The microorganisms were exposed to O₃ (ozone generator feeding by air) at concentrations ranging from 300 to 1500 ppm for 10 to 480 seconds with 18-20% RH: death rates in excess of 99.99% (> 4 log) were reached for both species at 1500 ppm O₃ and within 8 min.

[0006] Relying on much higher gaseous ozone concentrations, Held studied the efficacy of decontamination of hospital waste (Held B La generation d'O₃ a Ia pression atmospherique appliquee a Ia decontamination de surface 2002 Club Ecrin, Paris) on Gram-positive and Gram-negative vegetative bacteria, fungi, mycobacteria, and sporulated bacteria such as Clostridium perfringes, Bacillus atrophaeus and Geobacillus stearothermophilus. The decontamination vessel was supplied with ozone in a "cumulative mode", i.e. it is closed, allowing achieving a higher ozone concentration in this chamber, approximately 10000-12000 ppm, than is possible in a continuous flow with the given ozonator used. Each time waste is introduced, the chamber is replenished with fresh ozone, obtained from a dry-air corona discharge and, at the end of the cycle, the effluents released from the chamber are abated. Under these conditions, the system allowed inactivation of more than 10⁷ bacteria/mL (S. aureus, B. atrophaeus (formely B. subtilis), E. coli...) within an hour of exposure and more than 10⁷ spores/mL within two hours of treatment.

[0007] Ishizaki et al (J. Appl. Bacteriol. 60, 67-72, 1986) examined the sporicidal activity of gaseous ozone on different Bacillus spores with ozone concentrations ranging from 250 to 1500 ppm (0.5 to 3 mg/L), additionally focusing on the influence of the RH level. At RH levels of 50% or below, no appreciable decrease in the number of survivors was obtained after 6 h of exposure. However, at higher RH values, a 5 log reduction in less than 2 h was reached. Aydogan et al (J. Air Waste Manag. Assoc. 56, 179-185, 2006) results also showed that increasing the O₃ concentration from 1 to 3 mg/L with a 70-95% RH level increased the rate of inactivation of spores, but that beyond 3 mg/L (1500 ppm), only a weak additional increase was observed.

[0008] Two main points emerge from the above previous works: the higher the ozone concentration and the higher the RH (>50%), the more efficient is the inactivation process. As a matter of fact, in some cases, humidity is absolutely required to achieve sterility, as in the case of the TSO₃™ sterilization system, approved by both Health Canada and FDA (US Food and Drug Administration)). Furthermore, a few studies (see Kowalski et al, (Ozone ScL & Eng. 20, 205-221 , 1998) and Aydogan et al. (J. Air Waste Manag. Assoc. 56, 179-185, 2006) have additionally pointed out that the inactivation rates are far from being linear with increasing O₃ concentration, sometimes suggesting threshold levels: when O₃ concentration was increased, a tendency for saturation of the inactivation rate was experimentaly observed by these authors.

[0009] The processes, including the most recent ones, used to provide disinfection or antibacterial properties to surfaces are based on the implantation on these surfaces of active species known for their disinfectant properties such as bronopol, trichlosane, silver ions, Tiθ₂. It seems that the inactivation efficacy of such surfaces on microorganisms depends on the chemical nature of the molecule(s) implanted, on the homogeneity of the implantation treatement (spatial distribution), on microorganism number and on contact time.

SUMMARY OF THE DISCLOSURE

[0010] According to another aspect, there is provided a method of disinfecting and/or sterilizing at least one surface of at least one object. The method comprises exposing the at least one surface to dry ozone at a concentration of at least 500 ppm. The surface comprising at least one polymer.

[0011] It has been found that such a method can be effective for inactivating several types of microorganisms. As far as Applicants know, such an exposure to dry ozone has allowed observing for the first time, and in contrast to most of the published literature, that it is possible to inactivate bacterial spores under dry ozone conditions.

[0012] According to another aspect, there is provided a method for inactivating microorganisms. The method comprises exposing at least one surface of a polymer to dry ozone at a concentration of at least 50, 100, 150 or 200 ppm so as to convert at least a portion of the polymer into a biocidal polymer adapted to inactivate microorganisms:, and putting the microorganisms in contact with the at least one portion of the treated polymer

[0013] According to another aspect, there is provided a method for maintaining a polymer sterile for a given period of time. The method comprises exposing at least one surface of the polymer to dry ozone at a concentration of at least 50, 100, 150, or 200 ppm so as to convert at least a portion of the polymer into a biocidal polymer adapted to inactivate microorganisms contacting the at least one portion.

[0014] It has been found that when using the last two methods, surface(s) of a polymer treated with the method acquire(s) biocidal properties. Bacterial spores and vegetative bacteria are inactivated when deposited on surface(s) pre-treated according to the method. The methods have enabled the Applicants to achieve a new type of biocidal polymers.

[0015] According to another aspect, there is provided a biocidal polymer obtained by exposing at least one surface of the polymer to dry ozone at a concentration of at least 50, 100, 150, or 200 ppm.

[0016] According to another aspect, there is provided a biocidal material obtained by exposing at least one surface of the material to dry ozone at a concentration of at least 50, 100, 150, or 200 ppm.

[0017] According to another aspect, there is provided a biocidal polymer, the polymer having at least one surface that has been exposed to dry ozone at a concentration of at least 50, 100, 150, or 200 ppm.

[0018] According to another aspect, there is provided a biocidal polymer, wherein the polymer is effective for inactivating microorganisms over a period of at least 1 week.

[0019] It has been found that these polymers have biocidal properties and they can be used to inactivate microorganisms. Moreover, it was found that such polymers maintain over a considerable period of time such a biocidal effect.

[0020] According to another aspect, there is provided a method for inactivating microorganisms, the method comprising contacting the microorganisms with at least one biocidal polymer as defined in the present document.

[0021] According to another aspect, there is provided a kit comprising a biocidal polymer sealed in an air-tight container, wherein the biocidal polymer is effective for inactivating microorganisms over a period of at least 1 week and wherein the polymer comprises at least one dry ozone activated surface.

[0022] According to another aspect, there is provided a kit comprising a biocidal polymer sealed in an air-tight container, wherein the biocidal polymer is effective for inactivating microorganisms over a period of at least 1 week and wherein the polymer comprises at least one surface comprising carbonyl groups for example chosen from ester groups, carboxylic acid groups, aldehyde groups and mixtures thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In the appended drawings which represent various examples:

[0024] Figure 1 is a schematic view of an example of a device that can be used for carrying out methods as defined in the present disclosure; that part of the device within a dashed-line enclosure is used for humidified ozone processing.

[0025] Figure 2 is a schematic diagram showing the different steps of microorganisms recovery (1) after their exposure to the methods as defined in the present disclosure;

[0026] Figure 3 is a schematic representation of an example of an optical-absorption measurement system that can be used for studying spatial distribution of O₃ molecules (to check for spatial homogeneity) and for detecting species present in the sterilization/polymer activation chamber; gas flow is introduced along the x axis.

[0027] Figure 4a shows ultraviolet (UV) spectroscopy analysis

(transmitted UV intensity in an O₂ environment) of a sterilization chamber (with and without dry ozone) as shown in Figure 1 , and Figure 4b shows the absorbance plot for dry ozone corresponding to Figure 4a;

[0028] Figures 5a and 5b represent the measured optical absorbance at its maximum value (285 nm) transversely to the chamber of Figure 1 (z axis) at two different heights (y = 44.5 mm (mid-height), y = 52 mm (up)) as a function of axial distance from the chamber entrance (gas inlet location): Figure 5a in dry ozone; and Figure 5b in humidified ozone;

[0029] Figures 6a and 6b shows survival curves for 10⁶ G. stearothermophilus spores deposited on (untreated ozone) polystyrene (PS) Petri dishes after being treated in accordance with one example of a method as defined in the present document: Figure 6a concerning dry O₃ concentrations of 2000 ppmv and 4000 ppmv on "embedded" spores (spores embedded in bio-materials); Figure 6b concerning dry O₃ concentration of 4000 ppmv on "clean" spores; Figure 7 shows the effect of the incubation time on the number of Colony Forming Units (CFUs) recovered from 10⁶ "embedded" G. stearothermophilus spores deposited on polystyrene Petri dishes and exposed to an example of a method as defined in the present document i.e. dry gaseous ozone for 80 min at 4000 ppmv. A similar growth delay affecting only a small number of spores comparatively to the initial deposit, is also observed following exposure of "clean" G. stearothermophilus spores;

[0030] Figures 8a to 8d show SEM micrographs of "embedded" and

"clean" G. stearothermophilus spores (deposited on polystyrene Petri dishes): Figures 8a and 8c are for unexposed spores while in Figures 8b and 8d spores have been exposed to an example of a method in which the spores are exposed to dry ozone, as defined in the present document i.e. 4000 ppmv of dry O₃, for 80 min and 6 h, respectively; Direct dry ozone exposure of the spores does not affect significantly their morphology, as supported by the histograms in Figures 9(a) and 9(b).

[0031] Figures 9a and 9b represent statistical histograms of the length of G. stearothermophilus spores deposited on Petri dishes: Figure 9a unexposed "embedded" spores; Figure 9b "embedded" spores subjected for 80 min to 4000 ppmv of dry ozone; Figure 9c unexposed "clean" spores; Figure 9d "clean" spores subjected for 60 min to 4000 ppmv of dry ozone; these two exposure times, 60 and 80 min, corresponding or belonging to the second kinectic phase of survival curves presented in Figure 6 (Mahfoudh et al, 2010, Ozone: Sci. & Eng. 32, issue 3);

[0032] Figure 10 represents a survival curve for D. radiodurans vegetative bacteria deposited on polystyrene Petri dishes, dried and then subjected to an example of a method as defined in the present document, i.e. 4000 ppmv of dry gaseous ozone;

[0033] Figures 11a and 11b show a comparison of the survival curves of spores when treated in accordance to an example of a method as defined in the present document i.e. exposed to dry gaseous ozone at a 4000 ppmv concentration after being deposited : Figure 11a on glass Petri dishes; Figure 11b on polystyrene Petri dishes;

[0034] Figure 12 is a curve representing the number of B.atrophaeus spore survivors, expressed in log, as function of the contact time (including drying process) of a spore suspension, deposited on pre-treated polystyrene (PS) Petri dish surfaces, wherein the pre-treated PS illustrates an example of a biocidal polymer as defined in the present document;

[0035] Figure 13 is a curve, obtained from Figure 12, representing the inactivation rate of spores (expressed as a percentage of the number of deposited spores) as function of the contact time (including drying period) with the pre-treated surface (polystryrene Petri dish), wherein the spore suspension is in contact with an example of a biocidal polymer as defined in the present document;

[0036] Figure 14 is an histogram comparing the enzymatic activity of lyzozyme deposited on a glass Petri dish and on an example of a biocidal polymer (namely a treated PS Petri dish surface) according to the present document;

[0037] Figure 15 is an histogram showing contact angle measurements carried out, before and after dry ozone exposure, with distilled water on various examples of biocidal polymers according to the present document;

[0038] Figure 16 shows 3D atomic force microscopy (AFM) topographic images of (left) untreated and (right) ozone-treated polymer surfaces (60 min exposure to dry O₃ at 4000 ppm) according to the present document of: (a) PMMA, (b) PU, (c) PS and (d) silicone samples where full scale in the (x,y) plane is 5 μm;

[0039] Figure 17 shows 2D AFM topographic images (left) and corresponding phase images (right) of untreated (a) and treated (b) PMMA samples;

[0040] Figure 18 shows signals detected with a differential refractometer (DR) at the outlet of a chromatography column for an untreated and ozone-treated PS polymer exposed for 2h, as an example of the surface modification resulting from the ozone treatment, according to the present document;

[0041] Figure 19 shows a survival curve for ^β atrophaeus spores, expressed in percentage, that were deposited on a PS ozone treated polymer, according to the present document, in one case the ozone treatment time being 1 h and in the other case 3h, in both cases followed by a 1 h oven time;

[0042] Figure 20 shows the survival curve of Figure 19 expressed in log;

[0043] Figure 21 shows a survival curve for B atrophaeus spores, expressed in percentage, that were deposited on a PS ozone-treated polymer according to the present document; after 1 h exposure and vent time, in one case the PS ozone-treated Petri was stored in a hermetically closed (PE) bag while the other was left in ambient air;

[0044] Figure 22 shows the survival curve of Figure 21 expressed in log;

[0045] Figure 23 shows the percentage of carboxylic groups [0-C=O] detected on PS Petri dish surfaces as a function of the XPS spectra (x-ray Photoelectron Spectroscopy) of untreated, aged and recently ozone treated samples (60 min at 4000 ppm);

[0046] Figure 24 is an FTIR spectra (Fourier Transform Infrared

Spectroscopy) of an example of a biocidal polymer (PS Petri dish) according to the present document; ozone concentration was 4000 ppm;

[0047] Figure 25 is a graph showing the relative evolution of the concentration of the C=O bonds with treatment time taken from a biocidal polymer according to the present document;

[0048] Figure 26 is a graph showing the relative evolution of the concentration of the CHx (aliphatic and aromatic) bonds with treatment time taken from a biocidal polymer according to the present document; and

[0049] Figure 27 shows the percentage of the main chemical groups detected on PS Petri dish surfaces as obtained from XPS spectra (x-ray Photoelectron Spectroscopy) of untreated, aged and recently ozone treated samples (60 min at 4000 ppm).

DETAILED DESCRIPTION OF THE DISCLOSURE

[0050] The expression "dry ozone" as used herein refers, for example, to gaseous ozone with a relative humidity (RH) of less than about 2 % (determined with a hygrometer).

[0051] The expression "humidified ozone" as used herein refers, for example, to gaseous ozone with a relative humidity of more than about 2 %, generally about 50 % to about 80 % RH.

[0052] The term "disinfection" or "disinfecting" as used herein refers, for example, to inactivation of at least 3 log of microorganisms.

[0053] The term "higher disinfection" as used herein refers, for example, to inactivation of at least 4 log or 5 log of microorganisms on a total amount of about 10⁶ or more microorganisms.

[0054] The term "sterilization" or "sterilizing" as used herein means, for example, to inactivate at least 6 log of microorganisms on a total amount of 10⁶ or more microorganisms.

[0055] The term "inactivating" or the expression "to inactivate" as used herein refers, for example, to render the microorganisms unable to grow.

[0056] The expression "polymer maintaining its biocidal effect" as used herein refers to a polymer for which the number of survivors on a survival curve expressed in %, will not be increased by more than about 2 to 5%.

[0057] The expression "a biocidal effect having a persistence of at least

X days" refers to a biocidal effect which is not lowered by more than about 5 % over such a period of X days.

[0058] In the methods of the present disclosure and in the preparation of biocidal polymers, the concentration in ozone can be, for example, at least 200 ppm, 250 ppm, 400 ppm, 500 ppm, 750 ppm, 1000 ppm, 1500 ppm, 2000 ppm, or 2500 ppm. The concentration can also be about 1000 to about 10000 ppm, about 1000 to about 5000 ppm, or about 2000 to about 4000 ppm. [0059] For example, the at least one surface can be exposed to dry ozone for a period of time of at least 1 minutes, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes or 30 minutes.

[0060] For example, exposure to dry ozone is carried out over a period of time of about 30 to about 300 minutes, about 120 to about 240 minutes, about 150 to about 210 minutes, about 170 to about 190 minutes, about 30 to about 90 minutes, or about 45 to about 75 minutes. Even 6 h or 12 H of ozone exposure could be of interest for a longer remanent biocidal activity

[0061] For example, after the exposure to dry ozone, the treated polymer can be exposed passively to air or forced vented over a period of at least 30 minutes, at least 60 minutes, or at least 90 minutes.

[0062] For example, after the exposure to dry ozone the polymer can be exposed to an air jet. Such an exposure can be achieved for a period of time of at least 10, 15, 20, 30, 45 or 60 minutes.

[0063] For example, the exposure to dry ozone can be carried out over a period of time of about 30 to about 90 minutes or about 45 to about 75 minutes, and then the treated polymer is ventilated for a period of about 30 minutes to about 90 minutes or about 45 minutes to about 75 minutes.

[0064] For example, the dry ozone can have a relative humidity comprised between 0.1 and 1.9 %, 0.2 and 1.5 %, 0.3 and 1.2 %, or 0.4 and 1.1 %.

[0065] For example, the biocidal polymers can have a variation of contact angle of about 5 % to about 50 %, about 10 % to about 20 %, or about 25 % to about 45 % (measured with distilled water) as compared to the polymer prior to exposure to dry ozone.

[0066] For example, the at least one surface comprises carbonyl groups for example chosen from ester groups, carboxylic acid groups, aldehyde groups and mixtures thereof, present thereon that have been generated during the exposure to dry ozone. [0067] For example, the biocidal polymers can have a biocidal effect having a persistence of at least 5 days or at least 10 days. Alternatively, the biocidal polymers can have a biocidal effect having a persistence of about 7 to about 14 days.

[0068] For example, the at least one surface can be exposed to a dry ozone dose of at least 0.5 (min.%), 1 (min.%), 2 (min.%), 3 (min.%), 4 (min.%), or 5 (min.%). Alternatively, the dose can be about 1 (min.%) to about 48 min.%) or about 2 (min.%) to about 30 min.%).

[0069] For example, the at least one surface can be exposed to a dry ozone dose of at least 500, 1000, 2000, 3000, 4000, or 5000 ppm h. The dose can also be of about 500 to about 30000, about 1000 to about 24000, about 2000 to 20000, or about 2000 to 5000 ppm h.

[0070] According to one embodiment, the at least one object can be disposed into a reaction chamber and then, the at least one surface is exposed to a continuous flow of a gas comprising dry ozone.

[0071] For example, the gas can comprise at least 4 % of ozone, 6 % of ozone, 7 % of ozone, 8 % of ozone, or 10 % of ozone of the total gas flow. Alternatively, the gas can comprise about 2 % to about 8 % of ozone or about 4 % to about 8 % of ozone of the total gas flow. Care must be taken not to affect personnel when operating at such higher ozone concentrations: 4000 ppm represents 0.4% in O₂. For example, the gas can be at a temperature of about 20 to about 25 ⁰C.

[0072] According to one embodiment, the flow of gas can have a flow rate of at least 4 standard liter per minute (slm), 5 slm, or 6 slm.

[0073] According to another embodiment, the methods can further comprise analyzing and comparing UV absorption spectra, with and without ozone, of the chamber.

[0074] According to a further embodiment, the polymers can comprise polystyrene, high density (HD) polyethylene, polypropylene, polyurethane, silicone, polymethylacrylate (PMMA), styrene, or teflon. [0075] According to one embodiment, the polymer can comprise polystyrene or silicone.

[0076] For example, in the biocidal polymers, the least one surface effective for inactivating microorganisms can be a surface that has been dry- ozone activated.

[0077] For example, the polymers can be effective for inactivating microorganisms over a period of about 10 days to about 15 days.

[0078] For example, the at least one surface can comprise carbonyl groups (for example chosen from ester groups, carboxylic acid groups, aldehyde groups and mixtures thereof) present thereon.

[0079] For example, the polymers can be effective for inactivating microorganisms over of period of at least 5, 7, 10, 12, 14 or 15 days.

[0080] For example, the polymers can be effective for inactivating microorganisms over a period of about 1 to about 14 days, about 1 to about 10 days, about 1 to about 7 days, or about 1 to about 5 days.

[0081] For example, the polymers can be effective for inactivating microorganisms over of period of at least 2, 3 or 4 weeks.

[0082] For example, the polymers can be effective for inactivating by at least 2, 3, 4 or 5 log on an initial number of about 10⁶ microorganisms.

[0083] For example, the polymers can be effective for inactivating by at least 4 log a quantity of about 10⁶ microorganisms over a period of at least 2, 3, 4 or 5 days.

[0084] For example, the polymers can be effective for inactivating by at least 3 log a quantity of about 10⁶ microorganisms over a period of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 days.

[0085] For example, the polymers can be effective for inactivating by at least 3 log a quantity of about 10⁶ microorganisms over a period of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 days. [0086] For example, the polymers can be effective for inactivating by at least 2 log a quantity of about 10⁶ microorganisms over a period of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 days.

[0087] For example, the biocidal polymers of the present document can be sealed in an air-tight container. The container can be a film, a bag, a pouch etc. The container can be filled for example with ambient air or with an inert atmosphere (for example an inert gas such as argon, nitrogen etc.). The container can also be a sterilized container. For example, the container can comprise polyethylene. For example, the container can be a high density polyethylene bag.

[0088] For example, the biocidal polymer can be non-cytotoxic as determined in accordance with ANSI/AAMI/ISO 10993:2003 standard, part 5.

[0089] For example, the polymer can be chosen from polystyrene, HD polyethylene, polypropylene, polyurethane, silicone, polymethylacrylate, and Teflon.

[0090] For example, the polymers can maintain their biocidal effect on microorganisms over of period of at least 5, 7, 10, 12, 14 or 15 days.

[0091] For example, the polymers can maintain their biocidal effect over a period of about 1 to about 14 days, about 1 to about 10 days, about 1 to about 7 days, or about 1 to about 5 days.

[0092] For example, the polymers can be effective for inactivating microorganisms over of period of at least 2, 3 or 4 weeks.

[0093] For example, the methods and polymers of the present disclosure can be effective for inactivating by at least 4 log a quantity of about 10⁶ microorganisms present on the at least one surface.

[0094] For example, the methods and polymers of the present disclosure can be effective for inactivating by at least 5 log a quantity of about 10⁶ microorganisms present on the at least one surface.

[0095] For example, the biocidal polymers of the present disclosure can be effective for inactivating microorganisms over a period of at least one week, at least two weeks, or at least three weeks. [0096] For example, the methods of the present disclosure can comprise contacting the at least one portion of the surface with microorganisms, thereby inactivating at least a portion of the microorganisms.

[0097] For example, the methods of the present disclosure can comprise analyzing the microorganisms in order to determine the inactivation level of the microorganisms provided by the biocidal polymer.

[0098] The biocidal polymers prepared with the methods previously described can maintain sterility for a given period of time, which can be at least 1 week or at least 2 weeks. This given period of time can be at least 5 days, 10 days, or 15 days. For example, this given period of time can be about 5 days to about 10 days.

[0099] The following examples are presented in a non-limitative manner.

Examples

1 - Materials and methods

[00100] Due to its high oxidative power, ozone can damage various kinds of materials more or less severely. To minimize such possible effects, which could interfere with our experiments, the sterilization chamber, which is used as a processing chamber for treating polymers, is made from stainless steel (required for humidified ozone) and the windows used for spectroscopic observations are in fused silica. Inactivation of microorganisms deposited on Petri dishes that are either made from polystyrene or glass, or other kind of polymeric surfaces is investigated: (a) as deposited on such substrates without any pretreament and subjected to an ozone flow, (b) as deposited on such substrates that have been previously pretreated by an ozone flow.

1.1- Ozonation system

[00101] Figure 1 shows an example of a device that can be used for generating ozone and determine its concentration as it enters and exits the sterilization chamber. If desired, water vapour can be added to the ozone flow (system within the dashed-line enclosure in figure 1) and the relative humidity in the chamber measured with a hygrometer when a user wants to apply a humidified gaseous ozone treatment. The nature of the effluents exiting the chamber can be analyzed through Fourier Transform Infra Red (FTIR) spectroscopy. An ozone destructor (based on MgO, a chemical catalyser) is provided to abate ozone, whatever the experimental conditions, to release environment friendly gaseous by-products.

[00102] The preparation processing chamber can be a 400 mm long, 100 mm high and 220 mm wide parallelepiped (6 L volume). Since the ozone concentration cannot be monitored in the presence of humidified ozone with the analyzer (based on UV absorption), it is performed through FTIR spectroscopy by recording an O₃ signal close to 1000 cm^'1. The generated effluents can also be analyzed through FTIR spectroscopy.

[00103] The ozone generator operates with a corona discharge, supplied with dry gaseous O₂ (UHP grade) and can yield up to 6% gaseous O₃ comprised in a mixture of molecular and atomic oxygen in gas phase: decreasing the electrical current supplying the O₃ generator decreases the ozone concentration. Water vapour can optionally be added, as shown in Figure 1 (see the dotted-line enclosure representing such an optional feature), to the ozone flow, which otherwise is dry. When the sterilization chamber (Figure 1) is closed to begin a process, humid ambient air eventually present in it is flushed out by dry O2 gas. To achieve humidified ozone conditions, water is sent through a peristaltic pump to an "oven" (heater). The water vapour then produced is driven in the O₃ line by an incoming O₂ gas flow. The amount of water vapour injected, at a given temperature of the heater and given O₂ flow, depends on the H₂O flow set by the peristaltic pump. The corresponding relative humidity (RH) level in the chamber is determined with a hygrometer (Kahn) in the sole presence of O₂ (RH up to at least 95 % can be measured accurately (±0.3%)). The gas temperature in the chamber is close to ambient (« 22 ⁰C). Total gas flow is 5.64 and 2.6 standard liter/min (slm) under dry and humidified ozone conditions, respectively.

[00104] To comply with safety (toxicity) regulations, the system comprises at the end of the line (a) an ozone destructor which chemically transforms O₃ in O₂ and (b) a vacuum dry pump to make sure that effluents from the sterilization chamber are fully evacuated; also, under humidified ozone conditions, the chamber is located within a hood.

1.2- Microorganisms: choice of vegetative and sporulated bacteria for the study, their preparation for sample deposition and their number of survivors after exposure

[00105] To characterize the biocidal action of dry ozone, its effect on dried deposits of B. stearothermophilus endospores ATCC^® 7953 (renamed Geobacillus stearothermophilus), and on the bacterium Deinococcus radiodurans ATCC^® 13939 was examined. G. stearothermophilus endospores are routinely used in the validation of sterilization processes with wet heat. D. radiodurans bacteria, non-spore forming Gram-positive cocci, were used because of their resistance to radiation and dessication (needed feature to work with dried deposits similarly to those of spores).

1.3- Preparation of microorganisms

[00106] G. stearothermophilus bacterial spores, and D. radiodurans vegetative bacteria were prepared in the Laboratoire de contrόle des infections (Faculty of Dentistry) of Universite de Montreal. G. stearothermophilus vegetative bacteria were inoculated on a sporulation medium (Moreau et al 2000) and incubated for 3 days at 56-60 ⁰C. Spores were then collected, washed and stored at 4 ⁰C. Viability of the spores was determined by plating on Trypticase Soy Agar (TSA). D. radiodurans vegetative bacteria were collected after amplification in nutritive media at 3O⁰C for two days. Viability of the D. radiodurans bacteria was determined after deposition on control (unexposed) samples.

1.4- Sample preparation

[00107] Deposits of 10⁶ spores in 100 μl_ of sterile distilled water are made in the center of 60 mm diameter polystyrene or glass Petri dishes. As for D. radiodurans bacteria, they are added in the same fixed volume of 100 μl_; their number was sometimes much less than 10⁶ (sec. 3.1.4). The deposits are then dried out under ambient conditions and protected from light before being subjected to ozone treatment. [00108] Because O₃ interaction with glass is expected to be minimal (contrary to polystyrene), glass Petri dishes were used as the carrier material when testing O₃ inactivation efficacy under humidified ozone conditions, as is customary in such conditions (Ishizaki et al 1986 (J. Appl. Bacteriol. 60, 67- 72, 1986), Aydogan et al. 2006 (J. Air Waste Manag. Assoc. 56, 179-185, 2006).

1.5- Determination of the number of survivors after exposure.

[00109] Figure 2 shows the various steps involved in microorganism recovery. After exposure, 5 ml of 0.5% Tween™ in saline (15OmM NaCI) of saline is added to the Petri dish and microorganisms are released from its surface with mechanical scrubbing using a sterile swab. The recuperate is vortexed and serially diluted; after that, various volumes (50 to 200μL) of the different dilutions are spread onto Trypticase Soy agar plates. When viability is expected to be very low (less than 100 microorganisms), survivors are all collected through membrane filtration. The number of colony forming units (CFUs) is determined after various periods of incubation at specific temperatures: for G. stearothermophilus (56 ⁰C for 48h) and for D. radiodurans bacteria (ambient temperature for 4 days). Non exposed controls are recovered at the same time as exposed microorganisms. Specific germinants (aniline, dipicholinic acid/calcium, lysozyme) were used to ensure that permanent inactivation of exposed spores has been achieved.

1.6 Spatial distribution of the gaseous ozone concentration in the chamber through optical absorption spectroscopy

[00110] Figure 3 illustrates a schematic view of an optical-absorption measurement system, movable along the x-axis and possibly positioned at different heights y. The spectral source can be a deuterium lamp that provides significant continuum emission intensity in the 240-400 nm range. The fused silica window of the chamber extends axially on both sides of the chamber allowing visual observations and spectroscopy measurements. Axial distribution of the ozone concentration, at two different heights (y axis) in the chamber, i.e. at approximately !4 and ³A of the window height and for various axial positions (x axis), is obtained using an optical absorption spectroscopy system that can probe the chamber along the z axis, as schematized in Figure 3. A deuterium lamp is used as a continuum source of light, located at the focal length of a lens Li that transforms it into a parallel beam directed transversally to the chamber (z axis). On the other side of the chamber, at the same x and y positions, a diaphragm (Figure 3) admits only part of the light beam transmitted across the chamber, which is then focused with lens l_₂ on the input side of an optical fibre linked on its other extremity to the entrance slit of a 320 mm focal length spectrophotometer.

1.7- Ozone optical-absorption spectral characteristics

[00111] Figure 4a shows the intensity of a UV beam from a deuterium lamp after it has crossed the chamber transversally (along the z axis, Figure 3) as a function of wavelength, at mid-axial and mid-height positions (x = 200 mm, y = 44.5 mm, respectively), with and without dry ozone flowing in the chamber. The ozone molecules absorb the UV beam in a wide range of wavelengths (240 - 330 nm), as can be seen from Figure 4b that displays the corresponding absorbance curve of dry ozone. The maximum UV absorption is observed close to 285 nm.

1.8- Spatial uniformity of dry and humidified ozone concentrations

[00112] To determine the spatial uniformity of the ozone concentration in the chamber, absorption measurements at λ= 285 ± 0.5 nm (wavelength corresponding to the measured maximum absorption value for O₃ in dry ozone) were made as functions of axial position and at two heights y in the chamber. The results are shown in Figure 5, for both dry ozone (a) and humidified ozone (b), indicating an almost uniform distribution (within error bars) of the dry and humidified ozone concentrations in the chamber. This is supported by the fact that Applicants obtain the same inactivation rate of spores (taking into account the error bars) with Petri exposed at different axial positions in the chamber (not shown). The relatively low volume of the chamber, 6 L, most probably facilitates reaching homogeneity in the chamber. The humidified ozone concentration needs a few minutes to stabilize while, with dry ozone, concentration stability and homogeneity are reached more rapidly. 2- Exposure of microorganisms to dry ozone: experimental results and inactivation analysis

2.1- Gaseous dry ozone effect on microorganism viability and integrity of their structure

[00113] To gain insight into the inactivation mechanisms of the microorganisms subjected to gaseous ozone, Applicants are relying on the specific characteristics of the survival curves that they obtained and are also looking for eventual morphological changes of the microorganisms, using scanning electron microscopy (SEM) micrographs.

2.2- Geobacillus stearothermophilus spores survival curves

[00114] Generally speaking, the (scarce) literature discussing the exposure of spores to dry ozone mentions that these microorganisms are not, or very little, inactivated by such a treatment whereas subjecting them to humidified ozone (RH > 50-60%) eventually leads to their complete inactivation. This spore is a thermophile microorganism known to be resistant to humidified heat.

[00115] Experimental survival curves obtained for G. stearothermophilus spores are presented in Figures 6a and 6b. The essential difference between these two sets of data is that the spores are cleaner in Figure 6b (see Figures 8a to 8d), as a result of a more stringent washing procedure when preparing their suspension. In the case of Figure 6a, the spores are embedded in what could be bioproducts and/or chemical residues from the preparation of the spore suspension; it is interesting to compare these two cases since, at first, a thicker "coating" of the spore should delay the access of ozone to its inner parts, hence a slower inactivation rate. Nonetheless, in both cases, the inactivation efficiency of dry gaseous ozone on G. stearothermophilus spores is clearly strong. Applicants did not find indications in the literature concerning the inactivation of G. stearothermophilus spores when subjected to dry ozone conditions.

[00116] The survival curves in Figure 6a and 6b are characterized by a two-phase kinetics, the first phase having a much shorter D time. Recall that the second phase is believed to concern spores that are not directly accessible to the biocidal agent, in particular stacked spores, in contrast to the first-phase isolated spores and top ones on a stack (Boudam et al 2006, J. Phys. D: Appl. Phys. 39, 3494-3507). The dispersion of the data points on the survival curves with "embedded" spores (Figure 6a) is significantly larger than with clean spores (Figure 6b). Figure 6a also underlines that the higher the concentration of ozone , the higher the inactivation rate.

[00117] A peculiarity encountered when recovering G. stearothermophilus spores to work out survival curves concerns a longer incubation time needed for full growth of their CFUs on nutrient medium (see Figures 7a and 7b). Culture conditions of G stearothermophilus spores usually specify incubation for 48 h (at 56 ⁰C). However, as shown in Figures (7), it was observed that after 72 h the number of CFUs was more important than at 48 h; nonetheless, it was not much greater after a week, which was set as the (optimal) incubation time. Such a delay is known to occur when still viable spores are severely damaged. This phenomenon has been reported with spores exposed to oxidizing agents that have severe effects on their germination (Russel 2003 Antimicrobial Chemotherapy 52: 750-763, Cortezzo et al 2004, J. Appl. Microbiol. 97: 838-852).

2.3- Deinococcus radiodurans bacteria.

[00118] Because vegetative bacteria are less resistant to any given biocidal agent than sporulated bacteria, one expects them to have a comparatively much higher inactivation rate. Since our intent is to characterize as much as possible the inactivation of microorganisms under strict dry gaseous ozone conditions, Applicants needed a bacterium that could withstand its incorporation in a deposit to be dried, to keep the same experimental conditions as for spores and also to avoid water vapour from the suspension humidifing the gaseous ozone. Besides supporting desiccation, Deinococcus radiodurans is recognized as gamma (ionizing) radiation resistant and can also survive cold, vacuum, and acid. It is, in fact, known as a polyextremophile bacterium and has been listed as "the world's toughest bacterium". 2.4- Survival curves

[00119] Figure 10 shows a two-phase survival curve for D. radiodurans resulting from their exposure to 4000 ppmv of dry O₃. Clearly, dry ozone acting on this bacterium is much more efficient than with sporulated bacteria. Nonetheless, there is no mention in the literature of the possibility of inactivating dried vegetative bacteria with dry ozone: the ozonation process is reported to be efficient on vegetative bacteria in humid media, water, on agar or with airborne bacteria (Elford et al 1942, J. of Hygiene 42: 240-265, Kowalski et al 1998 Ozone Sci. & Eng. 20: 205-221)).

[00120] The number of D. radiodurans bacteria initially deposited and their eventual degree of stacking can vary considerably depending on culture and drying conditions. While for an initial deposit of 3.3x10⁴ bacteria (Figure 10), an average of 10 CFUs was counted after a 10 min exposure, in another independent experiment with an initial deposit of 5.9x10⁵ bacteria, only one CFU was encountered after the same exposure time.

2.5- Vegetative Bacteria and Sporulated Bacteria

[00121] The cell wall of the vegetative bacteria is essential for their growth, shape determination and resistance to environment fluctuation and aggression.

[00122] Spore forming Gram-positive bacilli, such as the Bacillus species, respond to stimuli such as nutrient deprivation by entering a state of dormancy, becoming endospores. Such a dormant state implies that the metabolic activity of the spore is greatly reduced, but that the spore retains the capacity to germinate back to the vegetative state. This is because the changes resulting from sporulation involve compartementation with new structures, namely core, cortex, plasma membrane, inner coat, outer coat and outer membrane. These structures confer resistance to several and different stresses. Besides common structural changes resulting from sporulation, there exist many differences among Bacillus species, which include their size, shape, resistance factors, water content and composition and organization of their chemical constituents. [00123] Knowing that O₃ must reach, remain onto and somehow penetrate and diffuse throughout the microorganism to damage it, its structural features can easily explain the much faster inactivation rate observed with the vegetative bacterium D. radiodurans (Di = 0.6 min) as compared with the sporulated G. stearothermophilus bacterium (Di =17 min).

[00124] Owing to its greater resistance to various types of stress such as dryness and because of its widespread use for the validation of sterilizing methods, B. atrophaeus (formely B subtilis) was used thereafter in Applicants' investigation of the biocidal efficiency of ozone-p retreated polymers.

2.6- Influence of the substrate nature on the kinetics of inactivation

[00125] Figures 11a and 11 b show inactivation kinetics of dried spores exposed to dry gaseous ozone. The inactivation rate is higher on polystyrene substrates (Figure 11(b)) than on Pyrex ones (Figure 11 (a)). On both types of substrates, the inactivation rate is significant and the survival curves are biphasic.

[00126] Applicants have shown that inactivation of microorganisms (deposited on PS Petri dishes) by exposure to gaseous ozone in dry case (RH < 2%) is possible. The presented results are markedly novel, implying that the literature in that respect, besides being scarce, must be reconsidered and updated.

[00127] Applicants have shown that inactivation (by gaseous ozone in dry case (RH < 2%)) of microorganisms deposited on other types of polymer is also possible as presented below.

[00128] Table 1 shows the inactivation efficacy after 60 min of dry ozone exposure of B. atrophaeus spores deposited on different polymer samples. Table 1

Inactivation efficiency %

Polymers

(S. atrophaeus spores)

Silicone 99.9985

Polyurethane 99.1

Polystyrene 79.7

High density polyethylene 36

Teflon (PTFE) 32.7

Polymethylmetacrylate 23.5

Polypropylene 15.2

3- Biocidal polymers, preparation and uses thereof

[00129] A standard Petri dish (made of polystyrene (PS)) was introduced, as is (from the factory), in the sterilization chamber of a device as shown in Figure 1. Prior to their exposure, the Petri dishes were immersed in a (10% diluted) cleaning solution (KOH and isopropyl alcohol: Patterson) and sonicated 5 min at ambient. They were afterwards rinsed 5 times with ultrapure and sterile water, and left to dry .After being treated with O₃, spores were deposited on it.

[00130] Later on, different types of polymer samples, some of them used in biomedical area, have also been tested.

[00131] Figure 1 shows the various elements utilized to generate ozone and determine its concentration as it enters and exits the chamber. The chamber, made from 316 stainless steel, is a parallelepiped 400 mm long, 100 mm high and 220 mm wide. Ozone concentration can be monitored with an analyzer based on UV absorption. The generated effluents can also be analyzed through FTIR spectroscopy. The ozone generator provides a mixture of molecular and atomic oxygen in the gas phase; it is operated in the electrical current range over which increasing the current increases the ozone concentration. The ozone flow is dry since the generator is supplied from (high-purity) O₂ dry-gas bottles. Total gas flow is set at 5.64 standard liter/min (slm) in order to achieve an ozone concentration of 4 000 ppm under our operating conditions. The gas temperature in the chamber is close to ambient (« 22 ⁰C).

[00132] An 0₃-destructor, localised at the end of the process line, is provided to abate ozone and to release it as O₂ to comply with safety (toxicity) regulations. Also for safety reasons, the chamber is located within a hood and a vacuum dry pump is used to make sure that the chamber effluents are fully evacuated at the end of the process.

3.1- Preparation of polymer surface and treatment conditions (dry ozome)

[00133] Different types of polymers (some used in the biomedical area) have been tested: polystyrene (PS) from Goodfellow® (ST313200), polyurethane (PU) from Johnston Industrial Plastics® (12348500), high density polyethylene (PE) from Goodfellow® (ET323100), polypropylene (PP) from Goodfellow® (PP303100), polymethylmetacrylate (PMMA) from Goodfellow® (ME303010), polymethydisiloxane (silicone) from Goodfellow® (SI303100) and polytetrafluoroethylene (Teflon®) from Goodfellow® (FP303050). Polymer sheets were cut up with appropriate techniques to provide samples of approximately 2.5 x 2.5 cm². Prior to their exposure, the coupons were immersed in a (10% diluted) cleaning solution (KOH and isopropyl alcohol: Patterson) and sonicated at ambient temperature. They were afterwards rinsed 5 times with ultrapure and sterile water, and left to dry. Each polymer sample was further laid out on a glass Petri dish, introduced in the processing chamber (Figure 1) and then exposed to ozone for 60 min (glass Petri dishes were used since the interaction of ozone with glass, unlike that with polymers, is expected to be minimal).

3.2- Microorganisms: sporulated bacterium choice, its preparation, deposition and recovery

[00134] The endospores used to test the biocidal properties of the ozone-treated polymeric surfaces were Bacillus atrophaeus ATCC^® 9372 (formely known as B. subtilis). These are routinely used in the validation of sterilization processes with dry heat, and are known to be very resistant to dry-ozone treatment. They were prepared in the Laboratoire de contrόle des infections (Faculty of Dentistry) of the Universite de Montreal.

[00135] Immediately after ozone treatment of the polymers, 10⁶ spores in 100 μl_ of distilled water were deposited at the center of the sample. These deposits were then left to dry, protected from light, for approximately 24 hours under ambient conditions (temperature and pressure) and then harvested. The whole microbiological protocol (preparation, deposition and determination of survivor number (recovery)) is the same as that described earlier by Mahfoudh et al. (Ozone: Science & Engineering. 32, issue 3, 2010).

3.3- Surface characterization techniques

3.3.1- Contact-angle measurements

[00136] The surface wettability of the polymers listed in section 2.2 was characterized before and after ozone treatment, using the water-drop-shape method (VCA Optima^® goniometer). Distilled water, glycerol and formamide were the three liquids used for contact-angle measurements. Two μl_ of each liquid were dropped onto 4 different sites on each sample to provide a statistical average for each sample. Experiments were conducted under ambient humidity and temperature conditions. From these contact-angle measurements using different liquids, one can calculate the surface energy.

3.3.2- Surface energy calculations

[00137] The surface energies are determined from the theories of Fowkes Ind. Eng. Chem 56 (1964) 40 (1) and Good J. Macromol. Sci. Chem. A26 (1989) 1183 (2), based on hydrophilic-hydrophobic interactions and on Lewis acid-base interactions, respectively,

Wsi = Yi (1 + cosΘ) = 2(γ,^d γ_s ^d)^1/2 +2(γ,^nd γ_s ^nd)^1/2 (1 ;

W₈, = Yi (1 + cosΘ) = 2(γ,^lwγ_s ^lw)^1/2 +2(Yi⁺Y_s ^")¹'² + 2(γfγ_s ⁺)^1/2 (Z where W is the work of adhesion between solid and liquid, and its components (dispersive γ^d, nondispersive (polar) γ^nd, Lifshitz-van der Waals γ^lw and acid-base γ⁺ γ^~); Y are the associated surface energies. 3.3.3- Atomic force microscopy (AFM)

[00138] AFM has been chosen to visualize the topologic morphology (degradation...) and to gather information on the surface roughness. AFM images were acquired in air at room temperature using a Digital Instruments (Dimension 3100, Santa Barbara, CA). Intermittent contact imaging, so-called "tapping mode", was achieved at a scan rate of 1 Hz using etched silicon cantilevers with a resonance frequency around 300 kHz, a spring constant of « 42 N/m, and a tip radius of <10 nm. All images were captured with a medium tip oscillation damping (20-30%).

3.3.4- Size exclusion chromatography (SEC)

[00139] This technique is used in the present case to look for any degraded or oxidized oligostyrene on the ozone-treated Petri-dish surface. The ozone-treated Petri dishes (exposure time of 10, 60 and 120 min) are rinsed with tetrahydrofuran (THF) solutions, leading to the dissolution of the O₃-treated top-layer; the resulting O₃-treated PS solutions are then injected in two sets of chromatography columns, corresponding to eluted high-molecular weight and low-molecular weight fractions. Both fractions are detected at the outlet of the chromatography column with a differential refractive index detector (differential refractometer (DR)) and UV detectors. The calibration of the elution volumes is run with different PS standards corresponding to molecular weight comprised between 3200 and 867 000 g.mol^"1.

[00140] With low-molecular weight fractions, the calibration curve is determined from different n-alkanes elutions. Therefore, the mass of eluted fractions of oxidized oligostyrene is given as n-alkane components.

[00141] After such a treatment, the exposed surfaces were found to be chemically modified, associated, at least for a certain time, with biocidal properties. Such biocidal properties remain for a given period of time and afterward decrease with time, even though some of the chemical modifications induced are permanent. It was observed that these time periods directly depend on the nature of the polymer. [00142] To demonstrate and observe the biocidal effect of a dry ozone- treated polystyrene Petri dish (see the experimental conditions previously described), various bacteria (E.coli, P.aeroginosa, S. aureus) and bacterial spores (B.subtilis, B.pumilus, G.stearothermophilus) have been deposited in PS-O₃- pre-treated dishes. After a given contact time (for vegetative bacteria 30 min and 3h), a nutritive media is deposited on these Petri dishes and, after incubation time, the efficacy of the treatment was visually observed. A nutritive media was added on treated surfaces after a given time of contact with microorganisms. Experiments show, for all vegetative bacteria (estimated number «10⁴), inactivation after 30 min of contact and no survivors after 3 hours of contact.

[00143] Table 2 shows the biocidal effect of a dry ozone-treated polystyrene Petri dish.

Table 2.

+ : positive growth: bacteria are viable even if a significant decrease of the number of survivors was noted.

- : negative growth: no bacteria were able to growth.

[00144] Bacterial spores were tested: about 10⁶ spores were deposited in 100μl_ of sterile distilled water on PS Petri dish surfaces treated according to the same method. When the deposits have dried, a nutritive media is added in each sample, then these are introduced into an incubator for about 24h. Note that the vegetative bacterium suspensions were never dried (to maintain them alive), consequently it can be considered that their surface interaction is less important than in the case of spore suspension deposit. Table 3 shows results obtained with the above-mentioned PS Petri dishes. Table 3.

Control Recently Aged samples pre-treated samples pre-treated samples

S. pumilus + - (n=4) (n=1) + (n=3 CFU=20 ± 18 3,

B. atrophaeus + - (π=4) + (n=4 CFIK100J

G. stearo + - (n=4) - (n=4)

+ microorganism growth (forming colonies)

- no microorganism growth n number of samples examined ft No survivors

[00145] Based on these results, it can be seen that (1) the treatment is quite efficient to provide a biocidal polymer and (2) that Petri dishes which were treated more than 1 month before deposition (aged samples) of microorganisms still presented a biocidal activity with a very good efficacy. Notice that these results were obtained after adding solid TSA nutritive media directly on surfaces.

[00146] Also, other control tests have been made after washing with water, alcohol and buffer solution on dry-ozone recently treated PS surfaces. The biocidal properties were not affected. It was observed: (1) that mechanical action on the surface (rubbing with water, alcohol, detergent, etc.) did not affect the biocide efficacy; (2) that some biocide efficacy remained for a period of time as long as one month. Moreover, water was added before nutritive media having in mind that spores may regenerated after re- hydratation, but no microorganism growth was observed. Note that all these observations were obtained with TSA nutritive media added directly on surfaces.

[00147] In order to make a comparative test, a PS surface was exposed to dry ozone during 60 minutes under the previously described conditions while another PS surface was exposed during 7 hours to UVC radiation (namely a mercury lamp generating a monochromatic emission at 254 nm (UVC lamp)). A control consisting of an untreated PS surface was also used. Then, B. atrophaeus (subtilis) spores were deposited on the three surfaces. After 24 hours of contact, no survivors were observed on the ozone treated surface. It was also observed that the UVC treated surface did not provide any biocidal effect as compared to the untreated surface. The amount of spores present on the UVC treated surface and the untreated surface was about the same.

[00148] A catalase was added on treated Petri dishes to determine whether H₂O₂ could be formed and adsorbed on the PS surface (after treatment), which could explain spores and bacteria death. This test shows that H₂O₂ is not readily involved in microorganism inactivation.

[00149] A dry ozone treatment of PS Petri dishes during only 10 min (with the same flow rate and concentration as previously mentioned) resulted in a less important biocidal activity .

[00150] The inactivation kinetics of pre-treated PS Petri dish surfaces on spores was studied as a function of contact time (see Figures 12 (survival curve) and 13 (inactivation rate curve)) The spores were initially in suspensions (10⁶ spores in 100 μl_ water) and evaluated during the course of the drying process (fully dried after 24 h).

[00151] Figure 13 shows the rapidity of the biocidal action of treated PS Petri dish surfaces on spores: 83.5% among them, from initialy 1.3 10⁶ spores, are dead after approximately a 3 hour contact (after 6h : inactivation rate is 97.8%; after 8h : inactivation rate is 99.91%; after 24h : inactivation rate is 99.9999% to 100%). The number of survivors decreases approximately exponentially with time as shown in Figure 12.

[00152] The biocidal polymer seems to denaturate proteins: this is inferred from the measurements of the enzymatic activity of the lyzozyme protein deposited on treated PS surfaces. 14μg of lyzozyme were deposited in 100μl_ of water on dry-ozone treated and untreated surfaces of PS and glass Petri dishes. Figure 14 represents the percentage of enzymatic activity remaining after contact times of 60 min, 3h and 6h of lyzozyme with PS surface exposed to O₃. Note that with direct exposure of lyzozyme protein to direct O3 flow (that is of a deposit on an untreated surface), it takes more than 3 hours to be able to detect a decrease in the lyzozyme enzymatic activity: this fact shows the specific and advantageous effects of the PS-treated surfaces. 3.4- Biocidal activity of tested polymers: inactivation efficiency

[00153] According to literature, surface modifications leading to antimicrobial properties generally imply coating or grafting of bio-active species (J. Sawai et al., Journal of Applied Microbioloy 96 (2004) 803; W. Zhang et al., Biomaterials, 27 (2006) 44.; and M. C. Yang et al. Journal of Polymer Research 9 (2002) 135). The tested microorganisms in such cases are mainly vegetative bacteria and viruses, while bacterial spores are rarely investigated (Sorensen et al., PCT Application published as W02008055501 (2008).

[00154] Makal et al. (Biomaterials 27 (2006) 1316) tested the biocidal efficiency of PU coated with chemical pendant groups on several Gram + and Gram - bacteria: inactivation of approximately 2.6 log, 3.3 log and 3.4 log of E.coli, P. aeruginosa and S. aureus were respectively obtained after a 30 min contact time (note that the bacterium suspension was sandwiched between two identically coated slides).

[00155] Table 4 presents the inactivation efficiency (in percentage) reached 24 hours after depositing S. atrophaeus spore suspension on ozone just-treated polymer samples.

Table 4.

Polymers ^Biocidal efficiency Deposit diameter

(%) on treated polymer (mm)

PS Petri dish 99.999 ±0.002 11 48 ± 2

Silicone 99.997 0.002 6-7 9 1

PS 99.7 0.2 8.5 34 1

PU 99.1 0.5 9 14 1

PP 15.2 4 8 12 3

PMMA 14.1 4 6 14 1

PE 10.5 13 7 22.9 0.2

Teflon 4.5 10 6 — 4 [00156] Percentage of inactivation efficiency observed 24 hours after depositing (10⁶/100μl) β. atrophaeus spore suspension on different polymer samples previously exposed 60 min to a 4000 ppm dry gaseous ozone flow. The deposit diameter D measured on each type of treated polymers is indicated. Percentage reduction in the contact angle of the untreated sample surface after its exposure to ozone is also displayed (distilled water was used for these measurements). Uncertainty on the measurements of the contact angle is typically ± 1 degree.

[00157] More particularly, Table 4 shows percentage of inactivation efficiency observed 24 hours after depositing (10⁶/100μl) B. atrophaeus spore suspension on different polymer samples previously exposed 60 min to a 4000 ppm dry gaseous ozone flow. The deposit diameter D measured on each type of treated polymers is indicated. Percentage reduction from the untreated-sample contact angle of the tested polymer samples after ozone exposure is also presented (distilled water was used for these measurements). Uncertainty on the measurements of the contact angle is in the ± 1 to ± 2 degree range.

[00158] A decrease in the number of survivors is, strictly speaking, observed for all samples, showing the ability to confer biocidal activity on these polymers. Teflon is known for its high chemical inertia (mainly because of C-F bonds), consequently modifications resulting from ozone exposure are expected to be lower than for other polymers.

[00159] The diameter D of the deposits (Table 4) is observed to vary from 6 up to 9 mm, implying that spore density can vary significantly according to the nature of the treated polymer. This affects the polymer surface interaction with spores. A possible consideration is that the larger D, the less stacked are the spores, implying a more direct contact with the treated surface and thus a higher inactivation efficacy. 3.5- Contact-angle modifications and biocidal actvity of tested polymers

[00160] The observed variation of the D values is expected to be related to the contact angle of the samples after their treatment. Figure 15 shows the contact-angle values determined before and after ozone treatment for each of the polymers considered, while Table 4 displays their percentage variation from the untreated value. Each ozone-treated sample shows some increase, small or large, of hydrophilicity (or wettability) depending on the polymer type.

[00161] Figure 15 is a graph showing various contact angle measurements determined with distilled water for a set of polymers before and after ozone treatment (4000 ppm, 60 min). Uncertainty bars are shown. Comparison of the biocidal efficiency with the contact angle variation (Table 4) leads us to claim that the variations in the inactivation rate and contact angle are not directly correlated. For instance, the contact-angle variation for treated PS and PE samples is 33.6% and 22.9%, respectively, but the corresponding inactivation efficiency is 99.7% and 10.5%. Moreover, the contact angle determined for treated silicone and PS samples is 110° and 56°, respectively, but both of them reached an inactivation rate of more than 99%. However, note that the highest inactivation rate was obtained with PS Petri dish samples Mahfoudh et a/. (Applied Surface Science, 256 3063-3072, 2010). that, among the treated polymers, yielded the lowest initial contact angle value and then the highest variation in the contact angle.

3.6- Physical characterization of the tested polymer surfaces

[00162] The observed hydrophilicity increase varies with the nature of the substrate: specific chemical interaction with the gaseous flow could possibly lead to different levels of physico-chemical modifications. Therefore, additional surface characterization of the polymers is needed to get more specific information on the physico-chemical modifications induced by ozone exposure. 3.7- AFM

[00163] Four polymers were analyzed with the AFM technique: silicone, PS and PU, which have a very high inactivation efficacy, while PMMA, has a very low one. The topographic modifications of these ozone-treated polymeric samples are presented in Figure 16.

[00164] Figure 16 shows 3D AFM topographic images of (left) untreated and (right) ozone-treated surfaces (60 min exposure to dry O3 at 4000 ppm) of: (a) PMMA, (b) PU, (c) PS and (d) silicone samples where full scale in the (x,y) plane is 5 μm. For all untreated samples the strong color contrast indicates that the gradient between the surface and the bottom of holes or top of peaks (reliefs) is very significant. The corresponding phase image for each untreated sample (not shown) indicates that there is only one chemical entity on the whole surface; however, for untreated silicone and PU samples a phase contrast is observed that is due to a great number of topographical variations.

[00165] The effect of ozone treatment on all these samples can be easily understood as a global abrasion of their surface, as seen in the 3D topographic images and in Table 5. It explains why the roughness (as measured by the root mean square (Rms) values of the topographic variations) generally decreases after treatment, the level of the decrease depending on the nature of the substrate. For instance, ozone treatment of PU samples leads to a great number of "canyons" of different depths, represented by more contrasted colors, although the sample roughness as a whole is very slightly affected. Hasirci et al. in High Performance Polymers 19, 621 2007 reported that after exposure to an oxygen plasma, the roughness values of PU samples increased slightly whereas in our conditions a very small decrease is observed. As for the treatment of PMMA samples, it leads to a widening of the already existing holes and to additional small holes such that the hole-free part of the surface covers only 76 % of the total surface (25 μm scale) as compared to 85 % before ozone exposure. Concerning the textured sample of silicone, the relief is clearly smoother after ozone treatment, representing a roughness reduction of 50% relative to the control sample; this trend is also observed with PS samples (an 80% reduction of the roughness after treatment). The diameter and depth of the holes on the treated PS sample were estimated to vary in the range of [1.2-3.4] μm and [12.6-46.1] nm, respectively. AFM analysis by Gejo et al. (Photochem. Photobiol. Sci. 5, 948 2006) of PS treated with VUV radiation in the presence of oxygen revealed the occurrence of 20 nm deep cavities, in agreement with our measurements.

[00166] Table 5 shows Rms values of polymeric samples before and after ozone exposure, as observed on a 5 μm (x,y) scale. Measurement uncertainty on typically four similar samples is ± 0.1 nm.

Table 5.

Rms values _{pMMA pu ps Si|jcone} (nm)

Untreated 6 .5 9.9 4. 8 > 60

Treated 4 .7 9.4 0. 9 32.7

[00167] White dots appear on the PMMA-treated 2D topographic images under 25 μm and also 5 μm scales. Such a white dot is identified by an arrow in the 2D topographical image in Figure 17(b)). These white dots are in reality peaks, as can be seen on the 3D topographic images (Figure 16) and are characterized by black dots on the corresponding phase (chemical contrast) images. Such peaks have been reported on certain polymeric surfaces after an oxidative treatment (see Mitchell et al., in Biomaterials 25, 4079, 2004, and Teare et al., in Langmuir 16 , 2818, 2000). Nonetheless, it appears that black dots are more numerous on the phase image than on the 2D topographic image, meaning that some peaks could be located slightly below the surface. As for the other kinds of polymer samples considered, no significant relative variations (before and after ozone exposure) have been observed; it is difficult to say whether or not new peaks are created after the oxidative treatment because of their small number density or because of the initially present peaks which could obscure these newly created typical peaks. 3.8- Surface energy calculated values inferred from contact-angle measurements

[00168] When using Fowkes's theory (Eq. (1)) to calculate surface energy, its value is assumed to be made of the sum of the dispersive and non-dispersive (polar) components of surface energy. It emerges from Table 6 that the total surface energy of all samples increases, to an extent that depends on the polymer nature. The increase of the surface energy implies that the surface has been converted from hydrophobic to hydrophilic groups (Wang et al., Surface and interface analysis, 37. 348, 2005).

[00169] No direct correlation between the biocidal efficiency and the variation of the non-dispersive components is observed in Table 6. As for the increase in hydrophilicity of the PS Petri-dish sample after ozone exposure, inferred from increased contact-angle values (Figure 15), it is easily related to the increase of the polar component of surface energy, suggesting in the present case that polar groups (known to increase wettability) could have been created on the surface following ozone treatment. This is actually the case since we have shown, in previous works using FTIR and XPS analysis (Mahfoudh et al. Ozone: Science & Engineering. 32, issue 3, 2010), that, after ozone exposure, a great number of chemical compounds containing polar bonds such as C-O, C=O were generated on the surface to the detriment of C-C bonds that did strongly decrease.

Table 6.

Polymers Biocidal efficiency Ynd Δγnd γto (%) (mJ.m^"2) (mJ.m^"2) (%) (mJ.rrr²)

C 9.28 34.62 43.90

PS dish 99.999^* 86.3 Tr 6.43 64.48 70.91

C 0.82 8.53 9.35

Silicone 99.997 89.8 Tr 0.02 16.19 16.21

C 16.60 8.01 24.61

PS 99.7 327.5 Tr 16.43 34.24 50.67

C 0.33 20.75 21.08

PU 99.1 -14.8 Tr 2.00 17.68 19.68

C 21.04 0.02 21.06

PP 15.2 44250.0 Tr 12.39 8.87 21.26 PMMA 14.1 C P R 6.?2?2 116fi.2 P¹5S _{1 m3} 2 ^2.47

r 4 ^n i *i λ4 19.64

PF m *i 45 8

^{Kt π υ D} Tr 9.73 22.37 ⁴⁰ ° 32.10

T fi si * ^{c 5}-^{62 5}-²⁶ _{ζ α} ^{1 0}-^{88 Teflon 4"5} Tr 6.07 4.95 ^{"5 9} 11.02

[00170] Table 6 shows the values of the dispersive and non-dispersive components of surface energy of the various polymers tested, as determined from Eq. (1): C (control) and Tr (after ozone treatment during 60 min at 4000 ppm). The sum of these two components is assumed to yield the total surface energy. The variation Δγ_nd of the non-dispersive component of surface energy after treatment relative to that of the control normalized to the control value is indicated in percentage.

[00171] Good's theory (Eq. (2)) used to calculate the surface energy is based on an acid-base model where the total surface energy is comprised of two components: γ^lw representing the Lifshitz-van der Waals interaction and γ^AB = 2(γ⁺ γ^')^1/2 modeling the acid-base interaction between the surface of the solid and that of the liquid, with γ⁺ and γ^~ being the corresponding acidic and basic components. These are displayed in Table 7.

Table 7.

Polymers Biocidal efficiency Δγ₊ Y- Δγ (%) (mJ ^Y.r⁺rr²2) (%) (mJ.rrϊ²) (%)

C 0.75 19.66

PS dish 99.999^* -53.3 119.0 Tr 0.35 43.06

C 2.31 3.55

Silicone 99.997 308.2 50.7 Tr 9.43 5.35

C 3.62 7.88

PS 99.7 -61.6 215.7 Tr 1.39 24.88

C 1.71 10.538

PU 99.1 -25.7 -13.5 Tr 1.27 9.12

C 12.70

PP 6.85

15.2 -87.6 9.6

Tr 1.58 7.51

PMMA 14.1 C 0.05 140.0 10.73 118.1 Tr 0.12 23.40

C 0 .13 9.07

PE 10.5 Tr 930 .8 1.34 16.82 85.4

C 0 .05 3.58

Teflon 4.5 Tr -20 .0 -6.4 0.04 3.35

[00172] Table 7 shows values of the acidic (+) and basic (-) components of the surface energy, as determined from Eq. (2) for the same group of polymers as in Table 6: C (control) and Tr (after ozone treatment during 60 min at 4000 ppm). Relative variations of these values normalized to the control value are given in percentages.

[00173] The relative variation of the basic component of the surface energy can be qualitatively correlated with the biocidal activity of the polymeric samples (Table 7). Table 7 shows that the basic component of the treated samples showing high biocidal efficiency has strongly increased, except for PU. The "bad behavior" of PU, to a less extent than that of silicone, could be related to the influence of hard and soft surface-segment repartitions (leading to hysteresis on the contact-angle measurement). Still for PU, other parameters such as roughness and the formation of weak boundary layers should also be taken into account. The above correlation is further supported by the fact that the Teflon surface, having the lowest biocidal efficacy, shows rather a decrease of its basic component after ozone exposure. The basic component could be associated to the formation of ester groups, the presence of which has already been reported by us (on Petri dishes) using FTIR spectroscopy and XPS analysis (see Mahfoudh et al. in Applied Surface Science, 256, 3063-3072, 2010).

3.9- Size exclusion chromatography analysis

[00174] To deepen the ozone-treated PS Petri dish analysis, a SEC analysis was conducted to identify and to quantify the presence of new chemical species characterized with molecules of different weights. 3.9.1- Analysis of the high-molecular weight fractions

[00175] Analysis of the high-molecular weight fractions indicates that degradation of the surface occurs following ozone treatement. Indeed, Table 8 shows that the weight-average molecular weight (Mw) and number-average molecular weight (Mn) have slightly decreased after ozone exposure. The polymolecularity index I=MwZMn has slightly increased after ozone exposure, implying that PS chains are being shortened by O3 treatment.

Table 8.

[00176] Table 8 shows weight-average molecular weight (Mw) and number-average molecular weight (Mn) and polymolecularity index (I) of the high mass fraction of the PS Petri-dish surface before and after ozone treatment (4000 ppm), at different exposure times.

3.9.2- Analysis of the low-molecular weight fractions

[00177] The SEC chromatograms of untreated and ozone-treated (120 min) samples are shown in Figure 18. SEC analysis confirms our previous experimental results suggesting the possible existence of a weak boundary layer (WBL): the presence of low-molecular weight fractions (ranging from 12 to 20 cm³) on the surface of both samples is clearly observable in the chromatograms. Relatively to the untreated sample, there are additional peaks, indicated by arrows, which correspond to degraded/oxidized products for which elution volumes are 13.88 and 14.86 cm³, respectively. The other peaks are identified as being associated to compounds added to the polymer, like stearate or naphtalate derivatives. [00178] Figure 18 shows signal detected with a differential refractometer (DR) at the exit of a chromatography column used for SEC analysis. It shows the variation, as a function of the elution volume, of the low-molecular weight fraction of PS Petri dishes exposed to 4000 ppm of ozone during 120 min and untreated ones (control). The arrows point at the molecular weight peaks generated by exposure of PS to ozone. J'aurais mis ce paragraphe avant Ie precedent?

[00179] From Figure 18, the new product proportions (from the peak area) can be estimated to be 7% for the first fraction (13.88 cm³) and 5 % for the second one (14.86 cm³). The 13.88 cm³ and 14.86 cm³ peaks were determined to correspond to molecules having 22 and 15 carbon atoms, respectively. These products could be identified as being dimers and trimers of the partially oxidized styrene (by considering the equivalence between the molecule structure and n-alkane calibration).

[00180] For elution volume of less than 12 cm³, the PS eluted fraction (not shown in Figure 18) of the treated sample highly decreases (by a factor of 1.8), confirming that enrichment in low weight products occurred on the surface.

[00181] The results presented previously demonstrated, after ozone exposure, the biocidal efficiency of various polymers. It was shown that upon ozone exposure, the polymers have a biocidal effect on various spores, and such an inactivation was related to physical and chemical properties of their surface, such as wettability and topography. Analysis of the surface energy of the polymeric samples revealed some correlation with the increase in the basic component of the surface energy after ozone treatment. Concerning the PS Petri dishes, chemical groups such as carbonyl groups (for example chosen from ester groups, carboxylic acid groups, aldehyde groups and mixtures thereof) (without being bound to such a theory) were identified as probably involved in the biocide activity. Chemical analysis (SEC) of the PS Petri dish surfaces demonstrated the presence of a slight degradation of the surface and enrichment in low-molecular weight products of the treated surface.

[00182] On practical grounds, it was demonstrated that some of these ozone-treated polymers can strongly reduce microorganism loads, eventually providing a high level of disinfection. Since polymers can be manufactured in many different forms (such as powders, films, plates,...), a large number of applications is conceivable, in particular in hospital environment. Various medical devices can thus me made using such biocidal polymers.

[00183] For example, the following results have been obtained 3.10- Analysis of the effect of the time of ozone treatment

[00184] Certain tests have been made to verify the influence of the time of treatment. In accordance with the procedure described above in sections 3 to 3.2, B.atrophaeus spores (10⁶/100μl) were deposited on ozone-pretreated PS Petri dishes. The Petri dishes have been treated, before deposition, under the same conditions as described in sections 3 to 3.2 and the compared exposure times were 60 minutes and 180 minutes. In all cases, after exposure to dry ozone, the Petri dishes have been ventilated under the fume hood for a period of 60 minutes. Such a ventilating step demonstrated, under certain circumstances superior results than when no such ventilation was carried out. Various depositions have been made after 0. 1 , 3, 14 and 31 days. Analysis of the deposits have been made as described in section 3.2. The results obtained are shown in Figures 19 and 20. Figure 19 shows a survival curve for B atrophaeus spores, expressed in percentage, which were deposited on a PS ozone treated polymer, in one case the ozone treatment time being 1 h and in the other case 3h, in both cases followed by a 1 h vent time according to the present document; Figure 20 shows the same on a log scale. Persistence of the biocidal effect is greater after 8 days for the samples pre- exposed 3 h instead of 1 h to the the dry ozone flow.

[00185] A longer exposure time to the ozone flow leads to a longer persistence of the biocidal effect of the polymers treated by dry ozone. 3.11- Analysis of the effect of maintaining the biocidal polymer in a sealed container

[00186] Some tests have been made in order to extend the biocidal effect of the polymer over time. In addition to the procedure described in sections 3.10, the so-obtained biocidal polymer has been sealed in an air-tight container. Such a container can be, for example, a film, a bag or a pouch made of various polymers. The biocidal polymer can be maintained under ambient air or in an inert atmosphere into the sealed container. The container can also be vacuumed. The container can be a sterilized bag. In such a manner, the biocidal polymer is packaged after being freshly prepared (and vented) so as to avoid prolonged contact with ambient air. A high density polyethylene internally sterilized bag (Bag Light™ sold by Interscience™) was used. The obtained biocidal polymer was inserted in the bag and the latter was sealed. Several samples have thus been prepared accordingly and their biocidal efficiency was tested, after a given number of elapsed days, by depositing B.atrophaeus spores (10⁶/100μl) thereon as described in section 3.9. In each case, the biocidal polymer has been withdrawn from the bag just before depositing the spores thereon. As it can be seen in Figures 21 and 22, maintaining the biocidal polymers in such an air-tight container allowed for extending the biocidal effect of the polymer over time. As it can be seen from Figure 21, a polymer treated with dry ozone over 1 hour and maintained in a polyethylene bag maintains its biocidal effect over a longer period of time than the same polymer which has not been inserted in such a bag. In fact, the results obtained with the polymer treated with dry ozone over 1 hour and maintained in the polyethylene bag are comparable to those obtained for a polymer that has been treated with dry ozone over 3 hours (see Figures 19 and 21 as well as Figures 20 and 22).

[00187] In addition to the results presented in Figure 20, some tests have been made with an 03 exposure of 1 hour without vent time. Various depositions have been made after 0. 1 , 3, 14 and 31 days. The results are shown in Table 9. Table 9.

3.12- Additional characterization of the surface of the biocidal polymer

[00188] The PS Petri dishes prepared by means of a dry ozone treatment as previously described have been further characterized by XPS (Figure 23 and 27), FTIR (Figures 24 to 26).

[00189] Figure 24 shows an FTIR spectra of untreated and treated samples in the 600-3600 cm^"1 range. Treated samples correspond to PS Petri dish surfaces exposed for 10 min and 60 min to dry gaseous ozone (4000 ppm). New peaks and a broad continuum appear respectively at around 1720- 1740 cm^'1 and 900-1300 cm^~1on 60 min treated surfaces. The presence of carbonyl groups such as esters groups, carboxylic acid groups, and aldehyde groups can be inferred from such results.

[00190] Figures 25 and 26 relates to the relative evolution of the concentration of the C=O and CHx (aliphatic and aromatic) bonds with treatment time, respectively. These results also support the presence of carbonyl groups such as esters groups, carboxylic acid groups, and aldehyde groups. Also, figure 26 shows degradation of the surface of the Petri dishes

[00191] Figure 27 (XPS) shows degradation from the surface of the Petri dishes. The various chemical groups present there also support the presence of the above-mentioned carbonyl groups.

3.13- Preparation of polymer surfaces and treatment conditions (humidified ozone)

[00192] It is also possible to render the surface of polymers biocidal by exposing it to a humidified O₃ flow. As an example, PS Petri dishes were used. Biocidal efficacy was tested by depositing on such treated surfaces B atrophaeus bacterial spores.

Table 10.

[00193] Table 10 shows comparative inactivation results obtained after having exposed the surface of PS polymers to dry ozone and humidified ozone for 60 min. The humidified ozone treatment has, within the error bar, the same efficiency as that of dry ozone. Considering a shorter ozone exposure time (10 min), the inactivation efficiency resulting from dry and humidified ozone exposure of PS Petri dishes led to 3083 ± 2..10⁴ (TSB negative) and 5176. ± 3.10³ survivors (2 out of 3 samples were TSB negative), suggesting a higher inactivation rate with humidified ozone exposure. In each case, 10⁶ spores were initially deposited in 100 μl_ of water and recovery process occurred after suspension drying.

[00194] Therefore, according to another aspect of the present document, there is provided a biocidal polymer obtained by exposing at least one surface of the polymer to humidified ozone, for example, at a concentration of at least 50, 100, 150, or 200 ppm.

[00195] According to still another aspect, there is provided a biocide polymer obtained by exposing at least one surface of the polymer to humidified ozone at a concentration of at least 50, 100, 150 or 200 ppm. According to yet another aspect, there is provided a biocide polymer obtained by exposing at least one surface of the polymer to ozone at a concentration of at least 50, 100, 150 or 200 ppm, wherein said ozone has a RH comprised between 2 % and 30 % or between 10 % and 20%.

[00196] The presence of water in the ozone flow generates specific chemical by-products such as, for example, the superoxide HO₂, a highly chemically reactive radical, which can interact with the surface of the polymer to be treated and possibly damage it to some extent. The concentration of these by-products increases with the RH. Using the lowest RH value practically achievable minimizes such interactions. Under certain circumstances, it is thus desirable to work with dry ozone so as to minimize possible damage to the polymer. This is particularly true when the polymer is to be treated more than once (providing more than once the biocidal effect) since being recycled. It would be possible to use ozone having a RH comprised between 2 % and 95 % but for some polymers, the treatment would probably be to harsh.

[00197] It can be seen that the biocidal effect is observed, to different extents, with the various kinds of polymers tested.

[00198] It can thus be mentioned that the present document describes novel biocidal polymers effective for inactivating various microoganisms (spores, bacteria...), including important densities of spores (10⁶/100μL).

[00199] The efficacy of the biocidal polymers was tested on spores because of their great resistance (15 times more resistant than vegetative bacteria).

[00200] The dry ozone pre-treatment previously mentioned can be carried out on various types of polymers largely used in hospital and industry environment. For example, it may be interesting to make use of this sterilizing or disinfecting effect on unpacked thermosensitive instruments a few days (2 or 3) before their use and, thanks to their remanent biocidal characteristics, keep them in the same degree of disinfection or sterility. Another possibility, more realistic, of the method is to sterilize bare thermosensitive instruments , then packed them for future use.

[00201] Disinfection may occur on liquid, humid or dry media. However the highest efficacy will be reached when contamination has occurred on a dry surface; surface effect efficacy is lower when contamination has occurred on a humid surface, but higher than for liquid media (and worse for large liquid volumes). The achieved efficacy will depend on the nature of the application medium and on the needed disinfection degree. [00202] It was observed that the biocidal polymers of the present document are non-cytotoxic as determined in accordance with ANSI/AAM I/ISO 10993:2003, part 5 requirements. These tests were conducted on PS ozone-treated .

[00203] It was also observed that it is possible to treat more than once a given polymer in order to render it biocidal. In fact it was observed that by treating the ozone activated surface by means of a plasma allows for cleaning the surface after it has been activated previously and preparing it for receiving a further treatment with ozone. It is also possible to treat more than once a given polymer without carrying out a plasma treatment, thereby saving time and costs.

[00204] Treatment of polymers allows to reach either good disinfection levels, and, in some cases and for certain polymers, sterility.

[00205] It must be underlined that when liquid nutrive media (namely TSB) is added to a spore deposit dried on a pre-treated PS Petri dishes, no growth is observed for as long as a week. It leads us to assume that the great majority of spores are killed as a result of their interaction with the pre-treated surface whereas others (a minority of them) are inhibited, i.e. cannot grow, as long as they remain in contact with this pre-treated surface. Consequently, when these inhibited microorganisms remain in contact with the pre-treated PS Petri dish surfaces, this surface can be considered as sterile. When TSA nutritive media is added, the "sterility" state (of the previously treated PS Petri dish surface, where spores were then deposited) is shown to maintain itself more than at least one month.

[00206] The treatment, thanks to its simplicity, can be carried out at low cost: it does not require expensive vacuum pumps, or biocidal agent "implantation" systems, it operates at atmospheric pressure and ambient temperature. Furthermore, no environment detrimental by-products are generated, as demonstrated through FTIR.

[00207] Generally, polymers can be produced in many different forms

(including powder, films, plates,...), so the pre-treatment of the method allows a great number of possible applications: for example glass coating in hospitals, specific clothes coating, package coating, bathroom furnitures, plates used in surgery room, pharmaceutical flasks, toys for immuno- suppressed children, etc. Several other applications are possible.

[00208] The treatment of the method could replace advantageously different kinds of surface biocidal coatings by active molecules because it is considerably less expensive. Moreover, in view of its efficacy on a great number of microorganisms and the remanence of this effect, furthermore since it does not require packaging during treatment, it renders it considerably promising and attracting.

[00209] The present disclosure has been described with regard to specific examples. The description was intended to help the understanding of the disclosure, rather than to limit its scope. It will be apparent to one skilled in the art that various modifications may be made to the disclosure without departing from the scope of the disclosure as described herein, and such modifications are intended to be covered by the present document.

Claims

CLAIMS:

1. A biocidal polymer obtained by exposing at least one surface of a polymer to dry ozone at a concentration of at least 50 ppm.

2. The polymer of claim 1 , wherein said concentration is at least 100 ppm.

3. The polymer of claim 1 , wherein said concentration is at least 150 ppm.

4. The polymer of claim 1 , wherein said concentration is at least 200 ppm.

5. The polymer of claim 1 , wherein said concentration is at least 1000 ppm.

6. The polymer of claim 1 , wherein said concentration is about 2000 to about 4000 ppm.

7. The polymer of claim 1 , wherein said concentration is about 1000 to about 5000 ppm.

8. The polymer of any one of claims 1 to 7, wherein said at least one surface is exposed to dry ozone for a period of time of at least 10 minutes.

9. The polymer of any one of claims 1 to 7, wherein said at least one surface is exposed to dry ozone for a period of time of at least 15 minutes.

10. The polymer of any one of claims 1 to 7, wherein said at least one surface is exposed to a dry ozone dose of at least 1 or 4 (min.%) and optionally of about 1 (min.%) to about 48 min.%) .

11. The polymer of any one of claims 1 to 7, wherein said at least one surface is exposed to a dry ozone dose of at least 1000 or 4000 ppm h.

12. The polymer of any one of claims 1 to 7, wherein said at least one surface is exposed to a dry ozone dose of about 2000 to about 5000 ppm h.

13. The polymer of any one of claims 1 to 12, wherein said biocidal polymer is obtained by disposing said polymer into a reaction chamber and, then, said at least one surface is exposed to a continuous flow of a gas comprising dry ozone.

14. The polymer of claim 13, wherein said gas comprises at least 4% of ozone of the total gas flow.

15. The polymer of claim 13, wherein said gas comprises at 6 % of ozone of the total gas flow.

16. The polymer of claim 13, wherein said gas comprises about 2 % to about 8 % of ozone of the total gas flow.

17. The polymer of any one of claims 13 to 16, wherein said gas is at a temperature of about 20 to about 25 ⁰C.

18. The polymer of any one of claims 13 to 17, wherein said flow of said gas has a flow rate of at least 4 slm.

19. The polymer of any one of claims 13 to 17, wherein said flow of said gas has a flow rate of at least 6 slm.

20. The polymer of any one of claims 13 to 17, wherein said flow of said gas has a flow rate of about 5 slm to about 6 slm.

21. The polymer of any one of claims 1 to 20, wherein said exposure to dry ozone is carried out over a period of time of about 15 to about 420 minutes.

22. The polymer of any one of claims 1 to 20, wherein said exposure to dry ozone is carried out over a period of time of about 30 to about 300 minutes.

23. The polymer of any one of claims 1 to 20, wherein said exposure to dry ozone is carried out over a period of time of about 120 to about 240 minutes.

24. The polymer of any one of claims 1 to 20, wherein said exposure to dry ozone over is carried out over a period of time of about 150 to about 210 minutes.

25. The polymer of any one of claims 1 to 20, wherein said exposure to dry ozone over is carried out over a period of time of about 170 to about 190 minutes.

26. The polymer of any one of claims 1 to 20, wherein said exposure to dry ozone over is carried out over a period of time of about 30 to about 90 minutes.

27. The polymer of any one of claims 1 to 20, wherein said exposure to dry ozone over is carried out over a period of time of about 45 to about 75 minutes. _/

28. The polymer of any one of claims 1 to 27, wherein after said exposure to dry ozone, said treated polymer is exposed to air over a period of at least 30 minutes.

29. The polymer of any one of claims 1 to 27, wherein after said exposure to dry ozone, said treated polymer is exposed to air over a period of at least 60 minutes.

30. The polymer of any one of claims 1 to 27, wherein after said exposure to dry ozone said polymer is exposed to an air jet.

31. The polymer of any one of claims 1 to 20, wherein said exposure to dry ozone over is carried out over a period of time of about 30 to about 90 minutes, and then said treated polymer is ventilated for a period of about 30 minutes to about 90 minutes.

32. The polymer of any one of claims 1 to 20, wherein said exposure to dry ozone over is carried out over a period of time of about 45 to about 75 minutes, and then said treated polymer is ventilated for a period of about 45 minutes to about 75 minutes.

33. The polymer of any one of claims 1 to 32, wherein said dry ozone has a relative humidity comprised between 0.1 and 1.9 %.

34. The polymer of any one of claims 1 to 32, wherein said dry ozone has a relative humidity comprised between 0.2 and 1.5 %.

35. The polymer of any one of claims 1 to 32, wherein said dry ozone has a relative humidity comprised between 0.3 and 1.2 %.

36. The polymer of any one of claims 1 to 35, wherein said biocidal polymer has a variation of contact angle of about 5 % to about 50 % as compared to said polymer prior to exposure to dry ozone.

37. The polymer of any one of claims 1 to 35, wherein said biocidal polymer has a variation of contact angle of about 10 % to about 20 % as compared to said polymer prior to exposure to dry ozone.

38. The polymer of any one of claims 1 to 35, wherein said biocidal polymer has a variation of contact angle of about 25 % to about 45 % as compared to said polymer prior to exposure to dry ozone.

39. The polymer of any one of claims 1 to 38 wherein said at least one surface comprises carbonyl groups present thereon that have been generated during said exposure to dry ozone.

40. The polymer of claim 39, wherein said carbonyl groups are chosen from ester groups, carboxylic acid groups, aldehyde groups and mixtures thereof.

41. The polymer of claim 39, wherein said carbonyl groups are ester groups.

42. The polymer of any one of claims 1 to 41 , wherein said biocidal polymer has a biocidal effect having a persistence of at least 5 days.

43. The polymer of any one of claims 1 to 41 , wherein said biocidal polymer has a biocidal effect having a persistence of at least 10 days.

44. The polymer of any one of claims 1 to 41 , wherein said biocidal polymer has a biocidal effect having a persistence of about 7 to about 14 days.

45. A biocidal polymer, wherein said polymer is effective for inactivating microorganisms over a period of at least 1 week.

46. The polymer of claim 45, wherein said at least one surface effective for inactivating microorganisms is a surface that has been dry ozone activated.

47. The polymer of claim 45, wherein said polymer is effective for inactivating microorganisms over a period of about 10 days to about 15 days.

48. The polymer of any one of claims 45 to 47, wherein said at least one surface comprises ester groups present thereon.

49. The polymer of any one of claims 1 to 48, wherein said polymer is effective for inactivating microorganisms over of period of at least 2 weeks.

50. The polymer of any one of claims 1 to 49, wherein said polymer is effective for inactivating by at least 2 log a quantity of about 10⁶ microorganisms.

51. The polymer of any one of claims 1 to 49, wherein said polymer is effective for inactivating by at least 3 log a quantity of about 10⁶ microorganisms.

52. The polymer of any one of claims 1 to 49, wherein said polymer is effective for inactivating by at least 4 log a quantity of about 10⁶ microorganisms.

53. The polymer of any one of claims 1 to 49, wherein said polymer is effective for inactivating by at least 5 log a quantity of about 10⁶ microorganisms.

54. The polymer of any one of claims 1 to 53, wherein said biocidal polymer is, once ozone treated, sealed in an air-tight container.

55. The polymer of any one of claims 1 to 54, wherein said biocidal polymer is non-cytotoxic as determined in accordance with ANSI/AAMI/ISO 10993:2003 standard, part 5.

56. The polymer of any one of claims 1 to 55, wherein said polymer is chosen from polystyrene, HD polyethylene, polypropylene, polyurethane, silicone, polymethylacrylate, and teflon.

57. The polymer of any one of claims 1 to 55, wherein said polymer is chosen from polystyrene, silicone, polyurethane, polymethylacrylate, and styrene.

58. The polymer of any one of claims 1 to 55, wherein said polymer comprises polystyrene or silicone.

59. The polymer of any one of claims 1 to 55, wherein said polymer is polystyrene.

60. A method for inactivating microorganisms, said method comprising contacting said microoroganisms with at least one biocidal polymer as defined in any one of claims 1 to 59.

61. A kit comprising a biocidal polymer sealed in an air-tight container, wherein said biocidal polymer is effective for inactivating microorganisms over a period of at least 1 week and wherein said polymer comprises at least one dry ozone activated surface.

62. A kit comprising a biocidal polymer sealed in an air-tight container, wherein said biocidal polymer is effective for inactivating microorganisms over a period of at least 1 week and wherein said polymer comprises at least one surface comprising ester groups.

63. A method rendering biocidal at least one surface of a polymer, said method comprising exposing said at least one surface to dry ozone at a concentration of at least 50 or 200 ppm over a period of time of at least 30 minutes.

64. The method of claim 63, wherein ester groups are generated at said at least one surface when exposing it to dry ozone

65. The method of claim 58, wherein said biocidal polymer has a variation of contact angle of about 5 % to about 50 % as compared to said polymer prior to exposure to dry ozone.

66. A method for maintaining a polymer sterile for a given period of time, said method comprising exposing at least one surface of said polymer to dry ozone at a concentration of at least 50 or 200 ppm so as to convert at least a portion of said polymer into a biocidal polymer adapted to inactivate microorganisms contacting said at least one portion.

67. The method of claim 66, wherein said method comprises contacting said at least one portion with microorganisms, thereby inactivating said microorganisms.

68. The method of claim 67, comprising analyzing said microorganisms in order to determine the inactivation level of said microorganisms provided by said biocidal polymer.

69. The method of any one of claims 66 to 68, wherein said given period of time is at least 1 week or at least 2 weeks

70. The method of any one of claims 66 to 69, wherein said given period of is about 5 days to about 10 days.

71. A method for inactivating microorganisms, said method comprising exposing at least one surface of a polymer to dry ozone at a concentration of at least 50 or 200 ppm so as to convert at least a portion of said polymer into a biocidal polymer adapted to inactivate microorganisms which have been unexposed to said dry ozone, and contacting said microorganisms with said at least one portion.

72. The method of claim 71 , wherein said microorganisms, once contacted with said portion, are at least substantially prevented from contacting ambient air.

73. A method of disinfecting and/or sterilizing at least one surface of at least one object, said method comprising exposing said at least one surface to dry ozone at a concentration of at least 500 ppm, said surface comprising at least one polymer.

74. The method of claim 73, wherein said concentration is at least 1000 ppm.

75. The method of claim 73, wherein said concentration is about 2000 to about 4000 ppm.

76. The method of claim 73, wherein said concentration is about 1000 to about 10000 ppm.

77. The method of claim 73, wherein said at least one surface is exposed to dry ozone for a period of time of at least 10 minutes.

78. The method of claim 73, wherein said at least one surface is exposed to dry ozone for a period of time of at least 30 minutes.

79. The method of claim 73, wherein said at least one surface is exposed to a dry ozone dose of at least 1 or 4 (min.%) and optionally of about 1 (min.%) to about 48 min.%) .

80. The method of claim 73, wherein said at least one surface is exposed to a dry ozone dose of at least 1000 or 4000 ppm h.

81. The method of claim 73, wherein said at least one surface is exposed to a dry ozone dose of about 2000 to about 5000 ppm h.

82. The method of any one of claims 73 to 81 , wherein said at least one object is disposed into a reaction chamber and then, said at least one surface is exposed to a continuous flow of a gas comprising dry ozone.

83. The method of claim 82, wherein said gas comprises at least 4 % of ozone of the total gas flow.

84. The method of claim 82, wherein said gas comprises at 6 % of ozone of the total gas flow.

85. The method of claim 82, wherein said gas comprises about 4 % to about 8 % of ozone of the total gas flow.

86. The method of any one of claims 82 to 85, wherein said gas is at a temperature of about 20 to about 25 ⁰C.

87. The method of any one of claims 82 to 85, wherein said flow of said gas has a flow rate of at least 4 slm.

88. The method of any one of claims 82 to 85, wherein said flow of said gas has a flow rate of at least 5 slm.

89. The method of any one of claims 82 to 85, wherein said flow of said gas has a flow rate of about 5 to about 6 slm.

90. The method of any one of claims 82 to 89, wherein said method comprises analyzing and comparing UV absorption spectra, with and without ozone, of said chamber.

91. The method of any one of claims 73 to 90, wherein said polymer is chosen from polystyrene, HD polyethylene, polypropylene, polyurethane, silicone, polymethylacrylate (PMMA), styrene, and teflon.

92. The method of any one of claims 73 to 90, wherein said polymer is chosen from polystyrene, silicone, polymethylacrylate (PMMA), and polyurethane.

93. The method of any one of claims 73 to 90, wherein said polymer is polystyrene.

94. The method of any one of claims 73 to 93, wherein said method is effective for inactivating by at least 4 log a quantity of about 10⁶ microorganisms present on said at least one surface.

95. The method of any one of claims 73 to 93, wherein said method is effective for inactivating by at least 5 log a quantity of about 10⁶ microorganisms present on said at least one surface.