ES3033109T3 - Reusable light-activated sterilization system for medical devices - Google Patents

Abstract

The present invention relates to an integrated, reusable photoactivated sterilization system, as well as a method for preparing the system. It also relates to a catheter plug that includes the reusable photoactivated sterilization system. (Automatic translation with Google Translate, no legal value)

Description

DESCRIPTION

Reusable light-activated sterilization system for medical devices

Technical field

The present invention relates to a reusable integrated light-activated sterilization system.

Additional aspects of the present invention include a medical device including the reusable light-activated sterilization system, and more particularly a catheter cap including the reusable light-activated sterilization system.

Background of the technique

Bacterial adhesion to surfaces is the starting point for biofilm formation and a difficult problem to address, compounded by the rise of drug-resistant bacteria, responsible for more than 500,000 deaths annually worldwide. Preventing bacterial adhesion and the subsequent biofilm formation is a demanding and costly task. However, it is of utmost importance in various contexts directly related to human health and life. Several problems can arise when these issues are not efficiently addressed, including microbe-induced corrosion, contamination of drinking water systems, oral diseases, biomedical implant failures, and several other issues directly related to public health. Providing surfaces with antibacterial properties has been a widely explored strategy. Conventional solutions have focused on the use of biocidal agents, such as antimicrobial nanoparticles or antibiotics, the latter remaining the primary weapon against bacterial infections. However, the overuse of antibiotics has contributed to the development of bacterial resistance, one of the greatest threats to public health today. Therefore, alternative strategies are needed to successfully address the problem. Photoactive bactericidal surfaces that destroy bacteria by using light combined with an appropriate photoactive agent rather than with biocides have attracted particular attention. Antimicrobial photothermal therapy (PTT) and photodynamic therapy (PDT) are two promising light-based methods. PTT is based on the irradiation of a photothermal agent that converts light into heat, leading to an increase in local temperature. As a consequence, membrane permeability increases, the expression of heat shock proteins is upregulated, and cellular components are denatured, ultimately leading to bacterial death. In PDT, irradiation and excitation with a nontoxic photosensitizer initiate the process of energy or electron transfer to oxygen molecules in the tissue, inducing the production of singlet oxygen (1O<2>) and/or other reactive oxygen species (ROS), respectively. This leads to the oxidation of various biological molecules, ultimately resulting in bacterial death. Unlike antibiotics, the development of resistance by bacterial cells is highly unlikely when using PTT or PDT.

Typically, photoactive bactericidal surfaces utilize different molecules, including gold nanomaterials, metallic nanoparticles, inorganic nanomaterials, and 2D materials such as graphene-based materials (GBMs), as well as combinations of these nanomaterials to achieve complementary performances. GBMs show great potential for use in these light-based therapies due to their high photothermal conversion efficiency combined with a remarkable broadband optical absorption capability arising from the closely spaced energy levels of the weakly held n-electrons.

However, the current state of the art on GBM systems is generally complex, as GBMs are combined with other materials or functionalized with organic molecules or serve as immobilization platforms for metal nanoparticles, antibiotics, or other photosensitizers. Moreover, these solutions generally rely on stimulation with lasers, which are expensive, have a small irradiation zone, and damage surrounding tissues, typically exceeding the skin irradiance tolerance limit established by the American National Standards Institute (ANSI) (~0.33 W/cm2 at 808 nm). Some examples are the combination of GO with red phosphorus, PHA polymers (polyhydroxyalkanoates), CuO nanowires, TiO<2> upconverting nanoparticles, or functionalization with aldehydes or other organic materials. Very frequently, these systems are solely or primarily based on the PTT effect. Efficient bacterial killing is generally achieved when temperatures reach above 100 °C, as in the case of the layer-by-layer GO/PHA film after irradiation with a NIR laser (1064 nm, 0.085 W) for 15 min. This, in turn, requires a significant temperature increase to achieve an effective antibacterial effect. These drawbacks compromise their translation to clinical or real-life applications, and most published work studies GBMs as suspensions in water rather than deposited on surfaces.

Systems comprising graphene-based layers and light sources are known from HUI LIWEIET AL.: “Surface Disinfection Enabled by a Layer-by-Layer Thin Film of Polyelectrolyte-Stabilized Reduced Graphene Oxide upon Solar Near-lnfrared lrradiation” and JP 2020081525 A.

Technical problem

Therefore, there remains a need for a sterilization system capable of providing an efficient antibacterial effect while remaining safe for human use. Furthermore, there is a demand for a reusable sterilization system that can be efficiently integrated into a medical device.

Summary of the invention

The present invention aims to solve the problems of the prior art by providing a reusable light-activated sterilization system according to claim 1.

It has been found that a sterilization system comprising a material having graphene and/or graphene oxide layers and a near-infrared light-emitting source (e.g., an LED source) as defined herein results in an efficient sterilization system. In these sterilization systems, the graphene and/or graphene oxide layers act as NIR-activated bactericidal platforms (photoactive surface) when irradiated with a safe, low-power NIR light-emitting source (e.g., light-emitting diodes (LEDs)).

Within the present disclosure, “sterilization system” means a system that has an antibacterial effect.

The sterilization systems according to the invention advantageously provide an antibacterial effect on both planktonic bacteria and adherent bacteria thanks to the combination of the NIR light source as defined above with the layers of graphene and/or graphene oxide.

When the present description refers to “preferred” embodiments/features, combinations of these preferred embodiments/features will also be considered as disclosed provided that this combination is technically significant.

Hereinafter, the term "comprising" should be understood as a non-limited disclosure, i.e., additional components or steps may be present or implemented, provided this is technically significant. For a more restricted embodiment, the term "consisting of" will be used and should be understood as a limited disclosure, i.e., without any additional components or steps.

Brief description of the figures

Figure 1: Antibacterial performance of the sterilization systems according to the invention against planktonic bacteria (S. aureus and S. epidermidis). Silicone films are used for comparison. FLG = few-layer graphene film, FLGO = few-layer graphene oxide film, SIL = silicone film, Bact = bacteria grown in the absence of any material. 3 replicates of each material in each independent test (n = 3).

Figure 2: Antibacterial performance of the sterilization systems according to the invention against adherent bacteria (S. aureus and S. epidermidis). The numbers at the top of the columns represent the percentage of dead bacteria. Silicone films were used as control material. #, o - statistically significant differences between samples with respect to total number (#) and dead adherent bacteria (o): one-way ANOVA to compare films within the same conditions (NIR off or NIR on) and unpaired t-test with Welch’s correction to compare the same film between NIR off and NIR on conditions. FLG = few-layer graphene film, FLGO = few-layer graphene oxide film, SIL = silicone film. 3 replicates of each material in each independent test (n = 3).

Figure 3: Physicochemical characteristics of FLG and FLGO films. (A) Surface morphology and (B) chemistry of the materials having graphene and/or graphene oxide layers as per the examples. Morphology and topography obtained by SEM (magnifications of 1000x and 5000x, scale bar = 50 |im and 10 |im, respectively). Atomic percentages of carbon (C 1s) and oxygen (O 1s) extracted from the scanning XPS spectra. The percentages of the chemical groups are shown in Figure 9. FLG = few-layer graphene film, FLGO = few-layer graphene oxide film.

Figure 4: Photothermal performance of FLG and FLGO films after 2 h incubation with growth media (TSB) followed by irradiation (NIR on) for 60 min (812 nm, 0.150 W/cm2). The NIR-induced temperature shift of films, surrounding supernatant, and surface was evaluated using a K-type thermocouple microprobe. FLG = few-layer graphene film, FLGO = few-layer graphene oxide film, SIL = silicon film.

Figure 5: Photodynamic properties of FLG and FLGO films. (A) Determination of glutathione (GSH) depletion by Ellman’s reagent and (B) reactive oxygen species (ROS) generation by FLG/FLGO films and by FLG/FLGO films in contact with bacteria (S. aureus and S. epidermidis) using the DCFH-DA probe, when samples were kept in the dark (NIR off) or irradiated (NIR on) for 45 min (812 nm, 0.150 W/cm2). (C) Schematic summarizing the possible pathways of oxidative stress onset. * Statistical significance, p < 0.05: one-way ANOVA comparing films under the same conditions (NIR off or NIR on) and unpaired t test with Welch’s correction comparing the same film between NIR off and NIR on conditions. * only indicates significant differences from its counterpart under NIR-off conditions. FLG = few-layer graphene film, FLGO = few-layer graphene oxide film, SIL = silicon film.

Figure 6: Mechanism of action of FLG and FLGO films towards bacteria when kept in the dark (NIR off, left side) and after irradiation (NIR on, right side). GSH = glutathione, GSSG = glutathione disulfide (oxidized glutathione), ROS = reactive oxygen species, PDT = photodynamic therapy, PTT = photothermal therapy, NIR = near infrared, FLG = few-layer graphene film, FLGo = few-layer graphene oxide film. The light grey colour in the representation indicates dead bacteria, the dark grey colour indicates live bacteria.

Figure 7: Adherent bacteria on FLG, FLGO, and SIL films. After 2 h of incubation with S. aureus and S. epidermidis, FLG and FLGO films were kept in the dark (NIR off) or irradiated (NIR on) for 45 min (812 nm, 0.150 W/cm2). Adherent bacteria were stained with SYTO9 (live) (grey spots) or PI (dead) (white spots), and images were obtained using an IN Cell Analyzer 2000 (scale bar = 10 |im).

Figure 8: Magnification of adherent bacteria on FLG, FLGO, and SIL films. After 2 h of incubation with S. aureus and S. epidermidis, FLG and FLGO films were kept in the dark (NIR off) or irradiated (NIR on) for 45 min (812 nm, 0.150 W/cm2). Adherent bacteria were stained with SYTO9 (live) (grey spots) or PI (dead) (white spots), and images were obtained using an IN Cell Analyzer 2000 (scale bar = 10 | im).

Figure 9: Chemical composition of surfaces when kept in the dark (NIR off) or irradiated (NIR on) for 45 min (Examples 1-2, Comparative Examples 1-2). Percentage of exposed functional groups extracted from PS scanning spectra: carbon double bonds (C=C), carbon single bonds (C-C), hydroxyl groups (C-OH), epoxy groups (C-O-C), carbonyl groups (C=O), carboxyl groups (O-C=O), and pi-pi (rc-rc) bonds.

Figure 10: Schematic representation of the antimicrobial assay procedure (Examples 1-2, Comparative Examples 1-3). After 2 h incubation of FLG and FLGO films with S. aureus or S. epidermidis to allow bacterial adhesion, samples were either kept in the dark (NIR off) or irradiated (NIR on, 812 nm, 0.150 W/cm2) for 45 min. Viability of adherent and planktonic bacteria was analyzed by LIVE/DEAD fluorescence assay and colony forming unit (CFU) and metabolic activity assays, respectively. The image depicting the NIR light source is not indicative of the positioning of the light source. In the examples, the NIR light source was positioned below the plastic well comprising the films.

Figure 11 A-E: Schematic representation of an embodiment of the sterilization system, i.e. the sterilization cap for a catheter.

Figures 12-13: Representation of the catheter cap according to the invention (prototype) and a catheter.

Figure 14: Prototype catheter cap.

Detailed description of the invention

The present invention discloses a reusable light-activated sterilization system comprising a material having layers of graphene and/or graphene oxide, and a near-infrared (NIR) radiation light-emitting source, preferably a near-infrared (NIR) radiation light-emitting diode (LED) source, wherein the light-emitting source emits radiation in the range of 650-950 nm with an intensity of 1-330 mW/cm2. A material having layers of graphene and/or graphene oxide according to the present invention is a film of graphene-based material (g Bm ) not combined with other photoactive materials, nor functionalized with polymers or organic molecules. In the present disclosure, this is also referred to as a “stand-alone GBM film”. In the present disclosure, this is also referred to as a GBM film.

The layers of the material comprising graphene and/or graphene oxide layers according to the invention may be graphene layers or graphene oxide layers, or combinations thereof. These two terms (graphene layers and graphene oxide layers) have the meaning commonly known in the field, i.e., graphene oxide refers to the oxidized form of graphene. Preferably, the graphene oxide as used herein has an oxidation state greater than 10%, greater than 20%, more preferably greater than 25%, for example, 30%.

The term “graphene material” (Gm) as used herein refers to a single sheet of carbon atoms packed together in a crystal lattice.

The term “few-layer graphene material” (FLGm) as used herein refers to a material having between 2 and about 5 graphene sheets, while the term “multi-layer graphene material” (MLGm) refers to stacks of between 6 and about 10 graphene sheets packed together.

The suffix “O” or “ox” is added when referring to the oxidized form, (e.g., FLGOm, GOm, MLGOm).

These graphene materials can be used to form the GBM films according to the invention, as described herein.

In the systems according to the invention, light-activated sterilization is due exclusively to the presence of the material having layers of graphene and/or graphene oxide, i.e., the graphene and/or graphene oxide layers, in combination with the irradiation of the light source as described herein. Therefore, the sterilization system according to the invention provides an efficient sterilization effect without the need for additional photoactive materials or other active components to enhance the effect.

The thickness of the material having layers of graphene and/or graphene oxide is not particularly limited, it may have a thickness of from 0.3 nm to 100 |im, preferably from 0.5 nm to 50 |im, more preferably from 100 nm-45 |im.

The material having layers of graphene and/or graphene oxide according to the invention may comprise from 2 to 800 layers of graphene and/or graphene oxide, preferably from 2 to 200, or from 2 to 100, more preferably from 2-20.

The material having layers of graphene and/or graphene oxide (independent GBM films) according to the present invention can be obtained by well-known techniques such as vacuum filtration, dip coating or spray coating.

For example, material containing layers of graphene and/or graphene oxide can be obtained using vacuum filtration. This is a simple technique that allows for obtaining GBM films that can be peeled from the filtration membrane and then used as stand-alone paper, or can be transferred onto an inert substrate. Film thickness can be controlled by adjusting the volume and concentration of the GBM suspension (e.g., FLGm, FLGOm, MLG/MLGOm, graphene) that is vacuum filtered, allowing the production of films ranging from a few layers to hundreds of layers.

The GBM suspension can be prepared using water or any other suitable organic solvent (e.g., ethanol, acetone, THF, 1-propanol, DMF, etc.). Preferably, the concentration of the GBM suspension is from 0.5 mg/ml to 80 mg/ml, more preferably from 1 mg/ml to 50 mg/ml.

It is evident from the content of the application that the material having layers of graphene and/or graphene oxide according to the invention (GBM film) may be a film having a flexible and stable structure that can be placed/transferred onto inert substrates to facilitate use/handling during sterilization, or it may be a film coated onto a substrate (a coating).

The coating can be obtained, for example, through dip or spray coating. For example, a suspension of GBM (e.g., FLGm, FLGOm, MLGOm, graphene) is prepared, and then the surface is immersed in the suspension to obtain a film of GMB (a material having layers of graphene and/or graphene oxide) coated on the surface. This technique can be used to deposit the material having layers of graphene and/or graphene oxide on various types of substrates without limitations.

The GBM suspension can be prepared using water or other suitable organic solvents (ethanol, acetone, THF, 1-propanol, DMF, etc.). Preferably, the concentration of the GBM suspension is from 0.5 mg/ml to 80 mg/ml, more preferably from 1 mg/ml to 50 mg/ml.

Examples of “material having layers of graphene and/or graphene oxide” that can be used in the present invention are graphene film, few-layer graphene film (FLG), few-layer graphene oxide film (FLGO), multi-layer graphene film (MLG), multi-layer graphene oxide film (MLGO), graphite, or combinations thereof.

In a preferred embodiment, the material having layers of graphene and/or graphene oxide is a “few-layer graphene” (FLG) film.

In another preferred embodiment, the material having layers of graphene and/or graphene oxide is a “few-layer graphene oxide” (FLGO) film.

In another preferred embodiment, the material having layers of graphene and/or graphene oxide is a “few-layer graphene and few-layer graphene oxide” (FLG/FLGO) film.

The FLG/FLGO films according to the present invention are arrays of few-layer graphene and/or few-layer graphene oxide. This means that an FLG/FLGO film may have several layers of graphene. These graphene layers are obtained by stacking few-layer graphene on top of each other to form a film or coating that may have a thickness in the nm or |im range.

The FLG/FLGO films according to the invention are obtained from FLGm or FLGOm, or combinations thereof. Therefore, at a microscopic level, it can be seen that the FLG/FLGO films are formed by several layers of FLGm/FLGOm.

Preferably, the thickness of the FLG/FLGO films according to the invention is from 0.3 nm to 100 |im, preferably from 0.5 nm to 50 |im, even more preferably from 100 nm-45 |im.

The FLG/FLGO films according to the invention may comprise from 2 to 800 layers of graphene and/or graphene oxide, preferably from 2 to 200, or from 2-100, more preferably from 2-20.

The FLG/FLGO films according to the invention may be a film that can be placed/transferred onto inert substrates to facilitate use/handling during sterilization, or they may be in the form of coatings, i.e. a film coated onto an inert substrate (a coating).

The sterilization system according to the invention additionally comprises a near-infrared (NIR) light emission source, preferably a light-emitting diode (LED). The near-infrared (NIR) light emission source (e.g., the light-emitting diode (LED)) according to the invention emits radiation in the range of 650-950 nm, preferably 700-900 nm, preferably 750-850 nm, preferably 800-820 nm, and most preferably 808-812 nm.

Furthermore, the near infrared (NIR) light emitting diode (LED) according to the invention emits NIR radiation with an intensity in the range of 1-330 mW/cm2, preferably 10-250 mW/cm2, more preferably 50-200 mW/cm2, for example, 150 mW/cm2.

Particularly preferred independent GBM films according to the invention are FLG and FLGO films, which in the present disclosure have been shown to be very effective even when stimulated with a safe low power energy source (650 nm-950 nm, 1-330 mW/cm2).

Accordingly, the present invention discloses a sterilization system comprising a material having layers of graphene and/or graphene oxide, also referred to as a free-standing GBM film, wherein this material acts as NIR-activated bactericidal platforms. Surprisingly, this free-standing GBM film can provide an antibacterial effect when used in combination with a safe, low-power NIR light-emitting source, preferably light-emitting diodes (LEDs).

Therefore, the sterilization systems according to the invention provide outstanding bactericidal action against planktonic and adherent bacteria. Specifically, almost complete elimination of bacteria in the supernatant (99%) and of adherent bacteria is achieved solely based on irradiation of the free-standing GBM film with a low-power NIR source. The results obtained were particularly good when the FLG and FLGO films were irradiated with a NIR-LED source according to the invention.

Surprisingly, the antibacterial effect of the sterilization systems disclosed herein is effective against both bacteria present in the supernatant (planktonic) and adherent bacteria. This is particularly advantageous for the elimination and/or prevention of biofilms, which are one of the main causes of infections, especially in the medical field. Consequently, the systems according to the invention can be used to sterilize medical devices. The sterilization systems according to the invention are particularly effective against gram-positive bacteria.

The material containing layers of graphene and/or graphene oxide is irradiated with a low-power NIR source (e.g., a NIR-LED source). This irradiation produces an antibacterial effect in the liquid surrounding the sterilization system and on the surface containing the material containing layers of graphene and/or graphene oxide.

Therefore, the sterilization system according to the invention can be integrated, incorporated, positioned in the liquid that needs to be sterilized to obtain an antibacterial effect.

The positioning of the light source relative to the material containing layers of graphene and/or graphene oxide is not particularly limited. It can be placed above the material containing layers of graphene and/or graphene oxide, or it can be placed below the material containing layers of graphene and/or graphene oxide. It is also possible to have a coating of the material containing layers of graphene and/or graphene oxide over the light source.

The irradiation period is not particularly limited and can be adjusted as needed. For example, the surface can be irradiated for at least 1 minute, preferably at least 10 minutes, or at least 20 minutes, or at least 45 minutes, or at least 60 minutes. In one embodiment, the radiation is emitted continuously for 1 to 60 minutes.

The irradiation time, in particular the maximum irradiation time, of the system to be sterilized is not particularly limited and may depend on the end application. For example, the sterilization system may be used to continuously irradiate the system to be sterilized throughout the day, or it may be used to provide intermittent irradiation repeated at regular intervals.

Without being bound by any theory, different mechanisms of action can be proposed for the sterilization systems according to the invention (Figure 6). Physical damage to the bacterial membrane has been mainly associated with the sharp edges of the free-standing GBMs of the invention (e.g., the sharp edges of FLG), which protrude from the surface. Furthermore, electron transfer and charge interaction can occur due to the conductive nature and presence of oxygen-containing groups on the free-standing GBMs (e.g., FLG and FLGo films), respectively, further contributing to the overall destabilization of the membrane. Considering the undetectable ROS production in the absence of light stimulation, physical damage associated with the intrinsic ability to cause GSH oxidation appears to be driving the observed antibacterial action, supporting an ROS-independent oxidative stress pathway.

Upon irradiation with NIR-LEDs, even though surface chemistry and morphology may weaken the bacterial membrane, the main contribution to the overall antibacterial activity comes from the photothermal (PTT) and photodynamic (PDT) action of the graphene-based materials. The photothermal properties of the material comprising graphene and/or graphene oxide layers, i.e., its ability to convert light into heat, causes the surface temperature to rise to a temperature between 45 °C and 60 °C, preferably between 50 °C and 57 °C. For example, a surface temperature of 51.3 °C can be obtained in the presence of FLG and a surface temperature of 56.0 °C can be obtained upon irradiation of FLGO films. The small temperature difference between the different materials comprising graphene and/or graphene oxide according to the invention suggests that the improvement in antibacterial performance could be mainly attributed to the photodynamic action. This is associated with the ability to induce ROS production, which together with GSH depletion, may greatly contribute to oxidative stress, since the degree of oxidative stress depends on competing actions of ROS generation and ROS scavenging reactions.

Based on the results of the DCFH-DA assay, the inventors surprisingly found that FLG films enhance ROS generation to a greater extent than FLGO films. Although oxidized materials are generally associated with increased ROS production due to a higher density of oxygen-containing functional groups, the conductive nature of the FLG films according to the invention may allow them to act as an electron pump (causing the disruption or oxidation of cellular structures without ROS production and through charge transfer from the extracellular membrane to graphene). Adsorption of ambient oxygen can still occur, but to a lesser extent.

The increased depletion of GSH by the free-standing graphene films according to the invention due to the increased oxidative potential upon irradiation greatly contributes to improved PDT efficiency, as the presence of GSH acts as a ROS scavenger. This may be particularly relevant in the case of FLGO films where GSH depletion may potentiate the effect of the lower ROS levels, implying that a predominant ROS-independent pathway for oxidative stress is taking place. Temperature may also be playing a more active role in the mechanism of action of irradiated FLGO films, considering the lower ROS production and increased bactericidal action. During irradiation, exposure to mild temperatures for at least 30 min may weaken the bacterial membrane structure, which, combined with ROS overproduction and GSH depletion, may allow excessive amounts of ROS to enter the bacteria more easily, contributing to the alteration of the intracellular redox balance. The singlet oxygen (1O<2>) produced can also enhance the permeability and thermal sensitivity of the bacterial cell membrane. Furthermore, the presence of ROS can also contribute to increasing the permeability of damaged bacterial membranes, making them more sensitive to heat and thus accelerating the leakage of intracellular contents from the bacteria. Finally, materials containing graphene and/or graphene oxide layers according to the invention maintain their surface chemistry and morphology. Consequently, the sterilization systems according to the invention can be used multiple times without degrading.

In the sterilization systems according to the invention, exposure of the material having graphene and/or graphene oxide layers to low intensity NIR-LED (650-950 nm with an intensity of 1-330 mW/cm2) dramatically improves its ability to kill planktonic (up to ~99%) and adherent (up to ~85%) methicillin-resistant S. aureus and S. epidermidis. Upon irradiation, a mild photothermal effect is observed in the supernatant, increasing the temperature from 37.0 °C to 39.0 °C-42.0 °C, while the surface temperature of materials having graphene layers (e.g., non-oxidized FLG films) increases up to 51.3 °C versus 56.0 °C for materials having graphene oxide layers (e.g., FLGO films). All tested materials promote full glutathione oxidation when irradiated, although FLG films induce higher ROS generation than FLGO films, suggesting that antioxidant depletion occurs preferentially via a ROS-dependent pathway (photodynamic effect) for FLG versus a ROS-independent pathway for FLGO films.

Therefore, the present invention demonstrates that safe, low-intensity NIR irradiation is a valuable and effective tool for enhancing the antibacterial performance of graphene surfaces through a synergistic photothermal and photodynamic effect. These free-standing NIR-activated graphene-based platforms (GBM films) therefore result in simple and cost-effective disinfection surfaces/systems, with widespread use in medical and non-medical applications.

Therefore, these sterilization systems can be applied in various healthcare applications where bacterial adhesion and infection are a concern (such as medical devices and implants), and in other non-medical applications that also require sterilization and disinfection (such as food packaging and water treatment).

For example, the use of a NIR-LED source with an irradiation intensity of 1-330 mW/cm2 makes these systems particularly useful for in vivo environments and for medical uses.

The independent GBM films according to the invention are coated on a waveguide connected to a low power emission source as described in the present invention, thereby resulting in an efficient integrated sterilization system that can be used to sterilize medical devices/surfaces/liquids.

The reusable light-activated sterilization system according to the invention further comprises a waveguide, which in this system is coated with said material having layers of graphene and/or graphene oxide. Preferably, the waveguide is connected to a NIR-LED source.

For example, in a preferred embodiment, the reusable light-activated sterilization system according to the invention further comprises an optical fiber, in which case the optical fiber is coated with said material having layers of graphene and/or graphene oxide. Preferably, the optical fiber is connected to a NIR-LED source. This integrated system can advantageously be inserted into the liquid to be sterilized, or within a lumen, for example, a catheter lumen, thereby resulting in an efficient integrated sterilization system that can be reused over time.

Catheter cap

Another aspect of the present invention relates to a medical device comprising the reusable light-activated sterilization system as described in the present disclosure. In particular, the sterilization system can be permanently or removably integrated into a medical device. For example, the sterilization system described herein can be integrated into a cap, thereby obtaining a sterilization cap. A preferred example of the sterilization cap according to the invention is a sterilization cap for a catheter as described herein.

A sterilization cap for a catheter comprising:

- a lid body that delimits a compartment;

- a power supply housed in said compartment;

- a light source connected to said power supply to emit near infrared (NIR) radiation in a range of 650 nm-950 nm with an intensity of 1-330 mW/cm2;

- a waveguide for said NIR radiation extending from the cover body and delimiting an outer surface;

characterized in that the outer surface of said waveguide comprises a material having layers of graphene and/or graphene oxide, preferably FLG and/or FLGO.

Preferably, said waveguide is at least partially coated with the material having layers of graphene and/or graphene oxide.

In the sterilization cap according to the invention, the cap body and the waveguide may define an annular gap between them for releasable insertion of said catheter. Furthermore, the sterilization cap may comprise complementary coupling means. Preferably, said complementary coupling means are positioned at least partially within said annular gap.

Preferably, said waveguide is inserted into a tubular portion of said lid body, said tubular portion terminating in said compartment on one side and terminating externally on a second, opposite side. The waveguide material of said waveguide is not particularly limited and may be any material that allows NIR radiation produced by the low-power emission source (e.g., by the LED source) to pass through. As an example, it could be a poly(methyl methacrylate) PMMA filament or a poly(ethylene terephthalate-glycol) (PETG) filament, as well as a glass filament. This waveguide may be at least partially coated with the material having layers of graphene and/or graphene oxide, these materials being as described in the present invention.

The method for partially incorporating the waveguide with the material having layers of graphene and/or graphene oxide according to the invention is not particularly limited. Various methods known in the art may be used; for example, coating the material can be obtained through dip coating. For example, a GBM suspension (e.g., FLG, FLGO, MLGO, graphene) is prepared, and then the surface is dipped into the suspension to obtain a GBM film (a material having layers of graphene and/or graphene oxide) on the waveguide.

The material having layers of graphene and/or graphene oxide used in the catheter cap is as described in the present application.

The near-infrared (NIR) light emitting source, preferably the light emitting diode (LED), is as described in the present disclosure. As already discussed, this LED source emits radiation in the range of 650-950 nm, preferably 700-900 nm, preferably 750-850 nm, preferably 800-820 nm and most preferably 808-812 nm.

In addition, the near-infrared (NIR) light-emitting diode (LED) emits NIR radiation with an intensity in the range of 1-330 mW/cm2, preferably 10-250 mW/cm2, more preferably 50-200 mW/cm2, for example, 150 mW/cm2.

Another embodiment of the present invention relates to a kit comprising:

- the sterilization cap as described herein, and

- a catheter that delimits a lumen into which said catheter cap waveguide can be at least partially inserted.

The catheter is not particularly limited; however, preferred types of catheters are hemodialysis catheters and chemotherapy catheters.

Experimental part

In the following experimental section, the antibacterial performance of the sterilization systems according to the invention against planktonic and adherent bacteria is evaluated. Exemplary sterilization systems are prepared using different graphene-based materials. In all examples, a low-power LED-based source with a maximum emission at 812 nm and an irradiance of 0.150 W/cm2 is used. The bacterial strains used are commercially available planktonic and adherent methicillin-resistant Staphylococcus aureus (ATCC® MRSA 33591™) and Staphylococcus epidermidis (ATCC® 35984™).

Preparation of bacterial cultures

Sterilization systems were challenged with methicillin-resistant Staphylococcus aureus (ATCC® MRSA 33591TM) and Staphylococcus epidermidis (ATCC® 35984TM). Bacteria were grown on tryptone soy agar (TSA, Merck) plates overnight at 37°C. Two colonies were picked, inoculated into 5 ml of tryptone soy broth (TSB, Merck), and grown overnight at 37°C with shaking at 150 rpm. Bacterial cultures were adjusted to 105-106 CFU/ml in freshly prepared TSB medium supplemented with 1% (v/v) human plasma to mimic the naturally occurring protein presence under physiological conditions.

Preparation of films consisting of graphene

Few-layer graphene (FLG) (~5 |im diameter, xGnP® grade M) was purchased commercially from XG Sciences (Lansing, USA) and oxidized to give few-layer graphene oxide (FLGO) using the modified Hummers method as described by Henrique et al., ACS Applied Materials Interfaces 12(18) (2020) 21020–21035. FLG powders were dispersed in water in an ultrasonic bath for 10 min, obtaining an aqueous FLG suspension. Both suspensions (FLG and FLGO) (45 mg in 10 ml) were vacuum filtered through a nylon membrane (pore size 0.20 µm, NY2004700, Merck) thereby obtaining FLG and FLGO films with a GBM mass/area distribution of 7.5 mg/cm2. The films were dried at 37 °C overnight and cut into 9 mm diameter discs (area 0.64 cm2) using a stainless steel punch. The FLG films were lightly compressed after production, for 5 s at ~16 kg/cm2, using a Graseby Specac press, to prevent particle detachment. All films were sterilized with ethylene oxide (EO gas, series 3+). In the following examples, few-layer graphene films and few-layer graphene oxide films are used as the graphene-based material according to the invention.

Example 1

FLG films prepared as above were transferred to 48-well suspension plates and incubated with 300 µl of bacterial suspensions (S. aureus and S. epidermidis) at 37 °C under static conditions. The remaining empty wells were filled with deionized water (dH<2>O) to prevent evaporation. Samples were incubated for two hours with S. aureus and S. epidermidis suspensions prepared as described above to allow bacterial adhesion. After the two-hour incubation, samples were irradiated for 45 min with an NIR LED (812 nm, 0.150 W/cm2) at room temperature (RT). The irradiation source was placed below the well plates, i.e., the irradiation first hits the plastic well and then the FLG films. The supernatant from each sample was then collected, and the surfaces were rinsed twice with fresh TSB to remove non-adherent bacteria (the wash media was also recovered and added to the supernatant). Viability of adherent and planktonic bacteria was analyzed using a LIVE/DEAD fluorescence assay and colony-forming unit (CFU) and metabolic activity assays, respectively.

Example 2

FLGO films prepared as mentioned above were transferred to 48-well suspension plates and incubated with 300 µl of bacterial suspensions (S. aureus and S. epidermidis) at 37 °C under static conditions. The remaining empty wells were filled with deionized water (dH<2>O) to prevent evaporation. The FLGO films were incubated for two hours with S. aureus and S. epidermidis suspensions to allow bacterial adhesion. After the two-hour incubation, the samples were irradiated for 45 min with the NIR LED (812 nm, 0.150 W/cm2) at room temperature (RT). The irradiation source was placed below the well plates, i.e., the irradiation first hits the plastic well and then the FLGO films. The supernatant from each sample was then collected, and the surfaces were rinsed twice with fresh TSB to remove non-adherent bacteria (the wash media was also recovered and added to the supernatant). Viability of adherent and planktonic bacteria was analyzed using a LIVE/DEAD fluorescence assay and colony-forming unit (CFU) and metabolic activity assays, as described below.

Comparative example 1

The samples were prepared in the same manner as in Example 1, except that they were not irradiated. The 45-minute waiting time was also maintained (no irradiation during this time).

Comparative example 2

The samples were prepared in the same manner as in Example 2, except that they were not irradiated. The 45-minute waiting time was also maintained (no irradiation during this time).

Comparative example 3

Silicone films, used as a comparative example, were transferred to 48-well suspension plates and incubated with 300 µl of bacterial suspensions (S. aureus and S. epidermidis) at 37 °C under static conditions. The remaining empty wells were filled with deionized water (dH<2>O) to prevent evaporation. The silicone films were incubated for two hours with S. aureus and S. epidermidis suspensions to allow bacterial adhesion. After the two-hour incubation, the samples were irradiated for 45 min with an NIR LED (812 nm, 0.150 W/cm2) at room temperature (RT). The irradiation source was placed below the well plates, i.e., the irradiation first hits the plastic well and then the silicone films. The supernatant from each sample was then collected, and the surfaces were rinsed twice with fresh TSB to remove non-adherent bacteria (the wash media was also recovered and added to the supernatant). Viability of adherent and planktonic bacteria was analyzed using a LIVE/DEAD fluorescence assay and colony-forming unit (CFU) and metabolic activity assays as described below. NIR irradiation of FLG and FLGO films

The FLG and FLGO films were kept in the dark (NIR off) (Comparative Examples 1-2) or exposed to NIR light (NIR on) (Examples 1-2 and Comparative Example 3). A low-power LED-based source with an emission maximum at 812 nm and an irradiance of 0.150 W/cm2 was used to irradiate the films. The irradiation source was positioned below the well plates; i.e., the irradiation first hits the plastic well and then the FLG films. In order to expose the bacteria to the maximum temperature for at least 30 min, 45 min was selected as the irradiation time for the antibacterial tests. In this way, the antibacterial activity was explored.

Characterization methods

The supernatant from each sample was collected (examples above), and the surfaces were rinsed twice with fresh TSB to remove non-adherent bacteria (the wash media was also recovered and added to the supernatant). Both the supernatant and the film surfaces were analyzed for planktonic and adherent bacteria, respectively, as described below.

Planktonic bacteria

The viability of planktonic bacteria was assessed by colony-forming units using the agar plate method. For this purpose, serial dilutions (10-1, 10'2, 10'3) of the supernatants were prepared and three 10 µl drops of each dilution were plated in TSA. The plates were incubated overnight at 37 °C and the CFU were counted. The viability of the bacteria was also confirmed by the alamarBlue™ assay. For this purpose, resazurin was added to the supernatants (10% v/v) and incubated for 2 h at 37 °C. The CFUs (Xex/Xem: 530 nm/590 nm) were measured on a microplate fluorometer (Spectra Max GeminiXS, Molecular Devices, California, USA) and correlated with the metabolic activity of the bacteria present in the medium. Eight replicates of each sample were evaluated (examples and comparative examples).

Adherent bacteria

The viability of adherent bacteria was determined by fluorescence staining using the LIVE/DEAD® BacLight™ Bacterial Viability Kit (L13152, Invitrogen). A mixture of SYTO9 nucleic acid dyes (Xex/Xem = 480/500 nm, green fluorescence) and propidium iodide (PI) (Xex/Xem = 490/635 nm, red fluorescence) was incubated with the samples for 15 min in the dark at RT. Samples were transferred to uncoated 24-well microplates (#82406, IBIDI, Germany) with the surface facing the glass bottom of the plate. Fluorescence images were acquired using an IN Cell Analyzer 2000 high-content detection microscope (GE Healthcare, Nikon 40x/0.95 NA Plan Apo objective) using a CCD camera (CoolSNAP K4). Three replicates of each sample were evaluated, acquiring 9 fields per sample (each field along a z-section of ~25 |im, with a z-step of 1 |im), with a total scan area of 1.44 mm2. The z-sections were projected onto a plane using IN Cell Investigator Developer Toolbox v.1.9.2 software (GE Healthcare). Bacterial quantification was performed as previously described by Henrique et al., ACS Applied Materials Interfaces 12(18) (2020) 21020-21035.

Impact of NIR irradiation on the physicochemical characteristics of the surface

The impact of NIR radiation on the morphology and surface chemistry (elemental composition and oxidation state) of the films was evaluated using scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS), respectively. The FLG/FLGO films were continuously irradiated for 45 min (812 nm, 0.150 W/cm2).

Scanning electron microscopy (SEM)

SEM analysis was performed using a Phenom XL SEM device in secondary electron detector (SED) mode and with an acceleration voltage of 10 kV. The FLG and FLGO films were bonded onto carbon tape and coated with a thin layer of Au by sputtering to enhance their conductivity.

X-ray photoelectron spectroscopy (XPS)

XPS analysis was performed using a Kratos Axis Ultra HSA (Kratos Analytical, UK) with a monochromatic Al X-ray source operating at 15 kV (90 W). Scanning spectra were acquired at 80 eV and high-resolution C 1s and O 1s spectra at 40 eV, with the C 1s peak referenced at 284.6 eV. Deconvolution of the high-resolution spectra was performed using CasaXPS version 2.3.16 processing software using a Shirley background. C 1s deconvolution was performed as previously described by Henrique et al., ACS Applied Materials Interfaces 12(18) (2020) 21020-21035.

Impact of NIR irradiation on photothermal properties

The light-to-heat conversion capacity of GBM films was also investigated after NIR irradiation. FLG/FLGO films were transferred to a 48-well suspension plate, immersed in 300 μl of TSB, and incubated for 2 h at 37 °C. After that, the samples were exposed to NIR light (812 nm, 0.150 W/cm2), and the temperature of both the surrounding medium and the film surface was recorded every 15 min for 60 min using a K-type thermocouple microprobe. Three replicates were used per condition, and two independent experiments were performed. Silicone films were used as a control surface.

Impact of NIR irradiation on photodynamic properties

Glutathione (GSH) levels, a key endogenous antioxidant, and reactive oxygen species (ROS) generation are two measures of oxidative stress in biological systems. Both can contribute to the photodynamic effect and were therefore quantified.

GSH oxidation assay

To determine GSH levels, GBM films were incubated with 300 µl of GSH (1.5 mM, Sigma) in reaction buffer (0.1 M sodium bicarbonate (pH 8), containing 1 mM EDTA) for 2 h at 37 °C. After incubation, samples were kept in the dark (NIR off) or NIR irradiated (NIR on) for 45 min (812 nm, 0.150 W/cm2). After that, 50 µl of the supernatant was mixed with 500 µl of the reaction buffer and 10 µl of Ellman’s reagent (10 mM in reaction buffer) for 15 min in the dark, allowing its reaction with the thiol groups of GSH to give fluorescent TNB. GSH was quantified spectrophotometrically at 412 nm and GSH leakage was calculated. GSH in reaction buffer without GBM films was used as a negative control, while GSH in H<2>O<2> (1 mM) was used as a positive oxidation control.

DCFH-DA assay

To assess ROS generation, GBM films were incubated alone or with bacteria for 2 h at 37 °C, followed by the addition of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA, ThermoFisher) at 10 µM. Samples were kept in the dark (NIR off) or NIR irradiated (NIR on) for 45 min (812 nm, 0.150 W/cm2). Fluorescence intensities were measured at 527 nm with excitation at 495 nm in a microplate fluorometer (Spectra Max GeminiXS, Molecular Devices, California, USA). Bacteria exposed to radiation were considered as initial activity and subtracted from the fluorescence intensities of bacterial films.

Results

The antibacterial performance of FLG and FLGO films against adherent planktonic methicillin-resistant S. aureus and S. epidermidis was evaluated after irradiation with a safe, low-power (0.150 W/cm2) near-infrared (NIR) (812 nm) diode (Examples 1 and 2). Compared to non-irradiated samples (NIR off) (Comparative Examples 1 and 2), the viability of planktonic bacteria was drastically reduced after 45 min of irradiation (NIR on). Nearly 90% of bacteria were eliminated in the presence of FLG films and almost 99% with FLGO films (achieving a 2-log reduction) (Figure 1), regardless of the bacterial strain. The results were consistent across both techniques used to assess bacterial viability, namely CFU counting and metabolic activity. Furthermore, no bacterial death was observed on silicone (comparative example 3), used as the irradiated control material, demonstrating that the antibacterial action was induced by the LED-NIR irradiated GBM film. Furthermore, the control bacterial culture showed no reduction in viability after irradiation, indicating that LED-NIR irradiation alone was safe for both bacterial strains.

Figure 1 shows the antibacterial effect of the sterilization systems of Examples 1 and 2 (FLG and FLGO films) on planktonic bacteria. After 2 h of incubation with S. aureus and S. epidermidis, the FLG and FLGO films were kept in the dark (NIR off) (Comparative Examples 1 and 2) or irradiated (NIR on) for 45 min (812 nm, 0.150 W/cm2) (Examples 1 and 2), the sample was prepared as described in the previous examples. Bacteria in the supernatant were evaluated by CFU counting and measurement of bacterial metabolic activity. Silicone films (Comparative Example 3) were used as control material. Bacteria grown in the absence of any material (Bact) were used as a control for the effect of irradiation on bacterial viability. * Statistical significance, p < 0.05: one-way ANOVA comparing films within the same conditions (NIR off or NIR on), and unpaired t test with Welch's correction comparing the same film between NIR off or NIR on conditions; * indicates significant differences relative to the NIR counterpart only.

As a result of NIR irradiation (NIR on), adherent bacteria in Examples 1 and 2 also showed an increase of up to 80% in percentage killing, and a significant reduction in adhesion, particularly in the presence of FLGO films (Example 2) and towards methicillin-resistant S. aureus (Figure 2, and Figures 7 and 8 for fluorescence microscopy images). In the absence of irradiation (NIR off) (Comparative Examples 1-2), methicillin-resistant S. aureus was already more susceptible to the action of FLG and FLGO films, with S. epidermidis showing a more pronounced increase in killing upon irradiation (NIR on). This is indicated by the enhanced uptake of propidium iodide (PI) dye which only enters membrane-compromised bacteria. No differences were observed on the silicone surfaces before and after irradiation (comparative example 3), validating the observed bactericidal effect of the sterilization systems according to the invention.

Figure 2 shows the antibacterial effect of the sterilization systems of Examples 1 and 2 (FLG and FLGO films) against adherent bacteria. After 2 h of incubation with S. aureus and S. epidermidis, the FLG and FLGO films were kept in the dark (NIR off) (Comparative Examples 1 and 2) or irradiated (NIR on) for 45 min (812 nm, 0.150 W/cm2) (Examples 1 and 2). Adherent bacteria were stained with SYTO9 (live) or PI (dead), visualized by fluorescence microscopy (Figure 7 and Figure 8), and counted.

Mechanism of action

Physicochemical characteristics of the surface

Figure 3 shows the physicochemical characteristics of FLG and FLGO films. (A) is the surface morphology and (B) is the chemistry of the films when kept in the dark (NIR off) and when irradiated (NIR on) for 45 min (812 nm, 0.150 W/cm2). Exposure to NIR irradiation for 45 min did not cause alterations in either the morphology (Figure 3.A) or the chemistry (Figure 3.B) of the surface of the FLG and FLGO films. The surface characteristics were similar under both conditions (NIR off and NIR on), with sharp edges protruding from the FLG films and wrinkles with similar morphology and size as those observed in the FLGO films. The oxidized materials exhibited high amounts of oxygen (~30%) and maintained the overall elemental composition after irradiation, with a carbon-to-oxygen (C/O) ratio of 28.4 (NIR-off) versus 26.75 (NIR-on) for FLG films, and 2.17 (NIR-off) versus 2.23 (NIR-on) for FLGO films. The percentages of chemical groups, and in particular oxygen-containing groups (hydroxyl groups (C-OH), epoxy groups (C-O-C), carbonyl groups (C=O), carboxyl groups (O-C=O)), were also similar (Figure 9), confirming that neither oxidation nor reduction was induced by the NIR-LED irradiation. In the absence of irradiation, the surface chemistry and morphology appear to contribute to the inherent bactericidal or anti-adhesive activity of the FLG and FLGO films. However, upon exposure to NIR radiation, something more than these characteristics per se is driving the overall mechanism of action.

Photothermal effect

The photothermal effect relies on a local temperature increase to kill bacteria. The photothermal properties of FLG and FLGO films were evaluated under conditions similar to the antibacterial assays (after 2 h of incubation in culture media at 37 °C) by assessing the supernatant and surface temperatures of the samples over 60 min of irradiation (Figure 4). In the presence of both FLG and FLGO films, the supernatant temperature increased during the first 30 min, remaining similar thereafter and within the range suitable for bacterial growth and viability (between 33 °C and 41 °C). However, the surface temperature showed an abrupt increase during the first 15 min, reaching 50 °C for FLG films and 55 °C for FLGO films. After 45 min of irradiation, temperatures reached 51.3 °C and 56 °C on the FLG and FLGO films, respectively, allowing adherent bacteria to be subjected to high temperatures for at least 30 min, thus enhancing the photothermally induced bactericidal effect. In the absence of the GBM films, the temperature of the growth media (TSB) remained below 37 °C, similar to when the silicone films were irradiated. This shows that LED-NIR alone is unable to induce a temperature increase, demonstrating the excellent photothermal efficiency derived from the GBM films, even at low irradiation intensities.

Photodynamic effect

The photodynamic effect relies on ROS production to kill bacteria. This increased ROS production disrupts the balance between ROS generation and scavenging, leading to oxidative stress. GSH, an endogenous antioxidant, is crucial for maintaining this balance; its depletion enhances the photodynamic effect by not scavenging ROS. The intrinsic oxidative potential of the FLG and<f>L<g>O films resulted in approximately 75% GSH oxidation (NIR off). Exposure to NIR irradiation (NIR on) resulted in a significant increase in GSH oxidation up to 95% (Figure 5.A), as indicated by a gradual fading from its typical yellow color due to the reaction between GSH and Ellman's reagent, which quantitatively translated into a decrease in absorbance at 412 nm. The silicone surfaces displayed negligible oxidative potential, regardless of NIR light irradiation.

Oxygen-containing groups on FLGO films can enhance GSH oxidation, as more reactive defect sites are available to adsorb water and O<2> molecules, increasing the formation of oxides and hydroxyl radicals on the surface. In addition to having reactive sites, the conductive nature of FLG films can also stimulate GSH oxidation, with FLG acting as an electron acceptor.

Photodynamic agents enhance ROS production. In the absence of NIR stimulation (NIR off), all graphene-based films elicited negligible amounts of DCF fluorescence, suggesting a lack of ROS in the presence of both GBM films and GBM films with bacteria (Figure 5B). Exposure of the films to NIR irradiation (NIR on) resulted in a significant increase in DCF fluorescence, indicating NIR-triggered ROS generation as a result of the association between the GBM films and NIR radiation. The silicone films showed residual levels of ROS production, regardless of the conditions. When GBM films are in the presence of bacteria, ROS levels are maintained regardless of the strain used. This suggests that either the bacteria are managing/balancing the overproduction of ROS, which is unlikely considering their high death rate, or the ROS molecules are interacting with the bacterial membrane, leading to ROS scavenging and bacterial damage or death. Based on both assays, an explanation for what is occurring can be hypothesized (Figure 5C). In the absence of NIR (NIR off), FLG and FLGO films actively contributed to GSH oxidation, however, they did not mediate ROS generation. On the other hand, radiation exposure (NIR on) increased the ability of FLG and FLGO to oxidize GSH (represented as thicker arrows), while also inducing ROS production. Despite similar GSH oxidation, FLG showed a significantly higher ability to induce ROS production than FLGO, thus suggesting ROS-mediated killing as the dominant factor in the antibacterial activity of FLG films. The reduced ability to induce ROS production indicates that FLGO films may act primarily through a ROS-independent pathway. The combination of both GSH oxidation and ROS production potentiates oxidative stress, due to the inability to neutralize and the resulting accumulation of ROS. This increases the effectiveness of the photodynamic effect, leading to a breakdown of cellular functions through DNA and vital enzyme damage and bacterial death.

Claims (15)

i. Reusable light-activated sterilization system comprising: - a material that has layers of graphene and/or graphene oxide, - a near-infrared (NIR) radiation light emission source, and - a waveguide, wherein the light emission source emits radiation in the range of 650-950 nm with an intensity of 1-330 mW/cm2, and wherein said waveguide is coated with the material having layers of graphene and/or graphene oxide. 2. The reusable light-activated sterilization system of claim 1, wherein the near-infrared (NIR) radiation light-emitting source is a light-emitting diode (LED). 3. Reusable light-activated sterilization system according to claim 1 or 2, wherein the light-activated sterilization is due to the presence of the graphene and/or graphene oxide layers. 4. Reusable light-activated sterilization system according to any of claims 1-3, wherein the material having layers of graphene and/or graphene oxide has up to 800 layers of graphene and/or graphene oxide. 5. Reusable light-activated sterilization system according to claim 1, wherein said waveguide is connected to the light emitting source. 6. Reusable light-activated sterilization system according to any of claims 1-4, wherein: the light emission source emits radiation in the range of 750-850 nm; and/or Radiation is emitted continuously for 1-60 minutes. 7. Reusable light-activated sterilization system according to any of claims 1-6, wherein the light emitting source emits radiation with an intensity of 10-250 mW/cm2. 8. Reusable light-activated sterilization system according to any of claims 1-7, wherein the material having layers of graphene and/or graphene oxide is: a few-layer graphene film; or a few-layer graphene oxide film. 9. A reusable light-activated sterilization system according to any of claims 1-7, wherein the material having layers of graphene and/or graphene oxide is a multi-layer graphene film and/or a multi-layer oxide film. 10. Reusable light-activated sterilization system according to any of claims 1-9, wherein the material having layers of graphene and/or graphene oxide has a thickness of 0.3 nm to 100 |im. 11. Medical device comprising the reusable light-activated sterilization system according to any of claims 1-10, optionally, wherein the medical device is: a catheter cap; or a catheter, preferably a hemodialysis catheter or a chemotherapy catheter. 12. Sterilization cap for a catheter comprising: - a lid body that delimits a compartment; - a power supply housed in said compartment; - a light source connected to said power supply to emit near infrared (NIR) radiation in a range of 650 nm-950 nm with an intensity of 1-330 mW/cm2; - a waveguide for said NIR radiation extending from the cover body and delimiting an outer surface; characterized in that the outer surface of said waveguide comprises a material having layers of graphene and/or graphene oxide. 13. Catheter cap according to claim 12, wherein said waveguide is at least partially coated with the material having layers of graphene and/or graphene oxide. 14. Catheter cap according to any of claims 12-13, wherein the material having layers of graphene and/or graphene oxide has up to 800 layers of graphene and/or graphene oxide. 15. Catheter cap according to any of claims 12-14, wherein the material having layers of graphene and/or graphene oxide is a few-layer graphene film or a few-layer graphene oxide film.