Patent Application
20240416143
This invention relates to methods and devices for improving healing of infection as well as healing behavior of a biological wound, kits for improving healing of infection as well as healing behavior of a biological wound, and methods for killing or inhibiting growth of microbes in a biofilm such as bacteria in a biofilm on or adjacent a biological wound.
Many types of large open wounds, such, as those associated with tissue grafts, burns, abrasions, ulcers and the like, require careful treatment to facilitate their closure and healing. A number of factors have been identified that promote healing of such wounds. These factors include, e.g., a sterile environment to avoid infection, an adequate blood supply to the wound region, sufficient oxygen and hydration, proper drainage, etc. Many types of wound dressings have been developed and studied for promoting healing of wounds.
In a study of wound infections in which antibiotics were antibiotic-susceptibility matched, treatment-induced resistance often resulted from rapid reinfection with a different strain, resistant to that antibiotic. The more prolonged and chronic the treatment, the more likely the development of that resistance. Complicating wound treatment is the fact that wound infections rapidly develop into biofilm infections, in which bacteria are encased in a matrix of bacterial carbohydrates, bacterial and host DNA, bacterial and host proteins, all further rendering biofilm-resident bacteria dramatically less sensitive to antibiotics.
Accordingly, there is a need to provide improved wound dressing devices and methods that provide one or more conditions conducive to healing of a biological wound.
The present invention meets the foregoing needs for improved healing of infection as well as wound dressing devices and methods that provide one or more conditions conducive to healing of a biological wound.
In one aspect, the disclosure provides a device for improving healing behavior of a biological wound. The device comprises: a matrix; a light-emitting layer comprising a plurality of light sources; a controller in electrical communication with the light sources, the controller being configured to execute a program stored in the controller to activate the light sources to irradiate the wound or a region adjacent the wound with light for a period of time when the device is placed over the wound; and an antimicrobial adjuvant present as part of the device in an amount effective to synergistically potentiate antimicrobial activity of the light with respect to microbes on or adjacent the wound.
In one embodiment, the controller is configured to execute the program stored in the controller to activate the light source to irradiate the wound or a region adjacent the wound with light for the period of time such that the light is provided at a radiant exposure of at least 50 J/cm2 during the period of time.
In one embodiment, the light has a wavelength that ranges from 380 nanometers to 700 nanometers. In one embodiment, the light has a wavelength that ranges from 380 nanometers to 410 nanometers.
In one embodiment, the plurality of light sources comprise an array of light emitting diodes in electrical communication with the controller, the controller being configured to execute a program stored in the controller to: (i) activate a first group of the light emitting diodes to irradiate the wound or the region adjacent the wound with light at a first range of wavelengths, and (ii) activate a second group of the light emitting diodes to irradiate the wound or the region adjacent the wound with light at a second range of wavelengths, the first range of wavelengths being different than the second range of wavelengths, and (iii) activate a third group of the light emitting diodes to irradiate the wound or the region adjacent the wound with light at a third range of wavelengths, the first range of wavelengths being different than the second range of wavelengths. In one embodiment, the first range of wavelengths ranges from 700 nanometers to 2000 nanometers, the second range of wavelengths ranges from 380 nanometers to 700 nanometers, and the third range of wavelengths ranges from 1000 nanometers to 1400 nanometers.
In one embodiment, the plurality of light sources comprise an array of light emitting diodes, and the light-emitting layer comprises at least one lens for focusing or dispersing light from at least a portion of the light emitting diodes.
In one embodiment, a translucent film positioned between the matrix and the light-emitting layer, wherein the film connects the matrix and the light-emitting layer.
In one embodiment, an antimicrobial adjuvant is present as part of the device in an amount effective to synergistically potentiate antimicrobial activity of the light with respect to microbes on or adjacent the wound.
In one embodiment, the antimicrobial adjuvant comprises a substituted naphthalene. In one embodiment, the antimicrobial adjuvant comprises an oxygen substituted naphthalene. In one embodiment, the antimicrobial adjuvant comprises 2-methyl-1,4-naphthoquinone. In one embodiment, the antimicrobial adjuvant is associated with the matrix.
In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of cells, drugs, precursors, enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, pharmaceuticals, photosensitizers, essential oils, therapeutics, and mixtures thereof. In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of tetracyclines. In one embodiment, the matrix includes an associated bioactive agent selected from minocycline, doxycycline, demeclocycline, and mixtures thereof.
In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of glycopeptide antibiotics.
In one embodiment, the controller is configured to execute the program stored in the controller to operate the light source using a plurality of cycles in which each cycle includes a first period of time in which the light source is on and a second period of time in which the light source is off. In one embodiment, the controller is configured to execute the program stored in the controller to operate the light sources using a plurality of cycles in which each cycle includes a first period of time in which a first group of the light sources is on and other groups of the light sources are off and a second period of time in which a second group of the light sources is on and other groups of the light sources are off. The first period of time can have a different length of time than the second period of time. The first period of time can have a same length of time as the second period of time.
The device can further comprise an oxygen sensor for sensing oxygen on or adjacent the wound, the oxygen sensor being in electrical communication with the controller. The device can further comprise a temperature sensor for sensing temperature on or adjacent the wound, the temperature sensor being in electrical communication with the controller. In one embodiment, the controller is configured to execute the program stored in the controller to turn off the light sources when the temperature sensor senses that the temperature has risen to a threshold temperature.
In one embodiment, the matrix has a surface including a channel, the surface facing the wound when the device is placed over the wound. The device can further comprise a source of irrigation fluid in fluid communication with an inlet of the channel. In one embodiment, the irrigation fluid comprises an additive. The additive can be selected from the group consisting of cells, drugs, precursors, enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, pharmaceuticals, photosensitizers, essential oils, therapeutics, and mixtures thereof. In one embodiment, the irrigation fluid comprises oxygen.
In one embodiment, the matrix has a surface including a plurality of channels, the surface facing the wound when the device is placed over the wound. In one embodiment, a source of irrigation fluid is in fluid communication with an inlet of the channels. The device can further comprise a waste collector in fluid communication with an outlet of the channels.
In one embodiment, the matrix is formed from a plurality of matrix components, each matrix component having a first section and a second section, the first section of one matrix component being dimensioned to matingly engage the second section of an adjacent matrix component to form all or a part of the matrix.
In one embodiment, a coolant fluid is in contact with the matrix or the light-emitting layer. In one embodiment, the matrix includes a cavity for receiving the coolant fluid. In one embodiment, the device further comprises a pump for recirculating the coolant fluid, the pump being in fluid communication with the cavity, the pump being in electrical communication with the controller which activates and deactivates the pump. In one embodiment, the device further comprises a container including a coolant fluid, wherein the container is in contact with the matrix or the light-emitting layer. In one embodiment, the coolant fluid is selected from biocompatible fluids. In one embodiment, the coolant fluid is a dielectric fluid.
The light emitting layer can comprise LEDs with one or more wavelengths for different purposes to be illuminated either sequentially or simultaneously. The range of wavelengths transmitted by the LEDs can range from 380-1300 nm, from 400-470 nm; from 600-700 nm, from 700-800 nm, from 800-1300 nm, comprising blue light, green light (500-600 nm), red light (600-700 nm), and near IR. The controller can be programmed to enable duty cycles ranging from 0% to 90% “on”; the matrix (e.g., bandage) layer can be relatively transparent enabling light transmission and either capable or not of being complexed with adjuvant antimicrobial substances. Wavelengths with or without additional antimicrobial substances can be tailored to attack either polymicrobial infections, gram-positive bacterial infections, gram-negative bacterial infections, anaerobic or aerobic infections, mycobacterial and fungal or mold infections.
In another aspect, the disclosure provides a device for improving healing behavior of a biological wound. The device comprises: a matrix dimensioned to cover the wound, the matrix having a surface including a channel, the surface facing the wound when the device is placed over the wound; a light-emitting layer comprising a plurality of light sources; a controller in electrical communication with the light sources, the controller being configured to execute a program stored in the controller to activate the light sources to irradiate the wound or a region adjacent the wound with light for a period of time when the device is placed over the wound; and a source of irrigation fluid in fluid communication with an inlet of the channel.
In one embodiment, the controller is configured to execute the program stored in the controller to activate the light source to irradiate the wound or a region adjacent the wound with light for the period of time such that the light is provided at a radiant exposure of at least 50 J/cm2 during the period of time.
In one embodiment, the light has a wavelength that ranges from 380 nanometers to 700 nanometers. In one embodiment, the light has a wavelength that ranges from 380 nanometers to 410 nanometers.
In one embodiment, the plurality of light sources comprise an array of light emitting diodes in electrical communication with the controller, the controller being configured to execute a program stored in the controller to: (i) activate a first group of the light emitting diodes to irradiate the wound or the region adjacent the wound with light at a first range of wavelengths, and (ii) activate a second group of the light emitting diodes to irradiate the wound or the region adjacent the wound with light at a second range of wavelengths, the first range of wavelengths being different than the second range of wavelengths, wherein the light at the first range of wavelengths is the antimicrobial adjuvant.
In one embodiment, the plurality of light sources comprise an array of light emitting diodes in electrical communication with the controller, the controller being configured to execute a program stored in the controller to: (i) activate a first group of the light emitting diodes to irradiate the wound or the region adjacent the wound with light at a first range of wavelengths, and (ii) activate a second group of the light emitting diodes to irradiate the wound or the region adjacent the wound with light at a second range of wavelengths, the first range of wavelengths being different than the second range of wavelengths. In one embodiment, the first range of wavelengths ranges from 600 nanometers to 2000 nanometers, and the second range of wavelengths ranges from 380 nanometers to 700 nanometers. In one embodiment, the controller is configured to execute the program stored in the controller to: (i) activate the first group of the light emitting diodes before activating the second group of the light emitting diodes.
In one embodiment, the plurality of light sources comprise an array of light emitting diodes in electrical communication with the controller, the controller being configured to execute a program stored in the controller to: (i) activate a first group of the light emitting diodes to irradiate the wound or the region adjacent the wound with light at a first range of wavelengths, and (ii) activate a second group of the light emitting diodes to irradiate the wound or the region adjacent the wound with light at a second range of wavelengths, the first range of wavelengths being different than the second range of wavelengths, and (iii) activate a third group of the light emitting diodes to irradiate the wound or the region adjacent the wound with light at a third range of wavelengths, the first range of wavelengths being different than the second range of wavelengths. In one embodiment, the first range of wavelengths ranges from 700 nanometers to 2000 nanometers, the second range of wavelengths ranges from 380 nanometers to 700 nanometers, and the third range of wavelengths ranges from 1200 nanometers to 1400 nanometers.
In one embodiment, the plurality of light sources comprise an array of light emitting diodes, and the light-emitting layer comprises at least one lens for focusing or dispersing light from at least a portion of the light emitting diodes.
In one embodiment, a translucent film positioned between the matrix and the light-emitting layer, wherein the film connects the matrix and the light-emitting layer.
In one embodiment, an antimicrobial adjuvant is present as part of the device in an amount effective to synergistically potentiate antimicrobial activity of the light with respect to microbes on or adjacent the wound.
In one embodiment, the antimicrobial adjuvant comprises a substituted naphthalene. In one embodiment, the antimicrobial adjuvant comprises an oxygen substituted naphthalene. In one embodiment, the antimicrobial adjuvant comprises 2-methyl-1,4-naphthoquinone. In one embodiment, the antimicrobial adjuvant is associated with the matrix.
In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of cells, drugs, precursors, enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, pharmaceuticals, photosensitizers, essential oils, therapeutics, and mixtures thereof. In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of tetracyclines. In one embodiment, the matrix includes an associated bioactive agent selected from minocycline, doxycycline, demeclocycline, and mixtures thereof.
In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of glycopeptide antibiotics.
In one embodiment, the controller is configured to execute the program stored in the controller to operate the light source using a plurality of cycles in which each cycle includes a first period of time in which the light source is on and a second period of time in which the light source is off. In one embodiment, the controller is configured to execute the program stored in the controller to operate the light sources using a plurality of cycles in which each cycle includes a first period of time in which a first group of the light sources is on and other groups of the light sources are off and a second period of time in which a second group of the light sources is on and other groups of the light sources are off. The first period of time can have a different length of time than the second period of time. The first period of time can have a same length of time as the second period of time.
The device can further comprise an oxygen sensor for sensing oxygen on or adjacent the wound, the oxygen sensor being in electrical communication with the controller. The device can further comprise a temperature sensor for sensing temperature on or adjacent the wound, the temperature sensor being in electrical communication with the controller. In one embodiment, the controller is configured to execute the program stored in the controller to turn off the light sources when the temperature sensor senses that the temperature has risen to a threshold temperature.
In one embodiment, the matrix has a surface including a channel, the surface facing the wound when the device is placed over the wound. The device can further comprise a source of irrigation fluid in fluid communication with an inlet of the channel. In one embodiment, the irrigation fluid comprises an additive. The additive can be selected from the group consisting of cells, drugs, precursors, enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, pharmaceuticals, photosensitizers, essential oils, therapeutics, and mixtures thereof. In one embodiment, the irrigation fluid comprises oxygen.
In one embodiment, the matrix has a surface including a plurality of channels, the surface facing the wound when the device is placed over the wound. In one embodiment, a source of irrigation fluid is in fluid communication with an inlet of the channels. The device can further comprise a waste collector in fluid communication with an outlet of the channels.
In one embodiment, the matrix is formed from a plurality of matrix components, each matrix component having a first section and a second section, the first section of one matrix component being dimensioned to matingly engage the second section of an adjacent matrix component to form all or a part of the matrix.
In one embodiment, a coolant fluid is in contact with the matrix or the light-emitting layer. In one embodiment, the matrix includes a cavity for receiving the coolant fluid. In one embodiment, the device further comprises a pump for recirculating the coolant fluid, the pump being in fluid communication with the cavity, the pump being in electrical communication with the controller which activates and deactivates the pump. In one embodiment, the device further comprises a container including a coolant fluid, wherein the container is in contact with the matrix or the light-emitting layer. In one embodiment, the coolant fluid is selected from biocompatible fluids. In one embodiment, the coolant fluid is a dielectric fluid.
The matrix (e.g., bandage) can contain a flexible plastic catheter enabling suction or irrigation of fluid. The addition of squeeze bulbs similar to Jackson-Pratt drains can be used to either irrigate, infuse, or suck out wound substances to further enable healing and mitigate microbial infection. The catheter inserted into the matrix can also be connected to mechanical pumps for suction or infusion.
In another aspect, the disclosure provides a kit for improving healing behavior of a biological wound. The kit comprises: a matrix dimensioned to cover the wound; at least two light-emitting layers, each light-emitting layer comprising a plurality of light sources, at least one of the light-emitting layers being configured to irradiate the wound or a region adjacent the wound with light at a first range of wavelengths, and at least another of the light-emitting layers being configured to irradiate the wound or the region adjacent the wound with light at a second range of wavelengths, the first range of wavelengths being different than the second range of wavelengths; and a controller configured to be placed in electrical communication with the light sources of each light-emitting layer, the controller being configured to execute a program stored in the controller to activate the light sources of each light-emitting layer to irradiate the wound or the region adjacent the wound when the device is placed over the wound.
In one embodiment, the kit further comprises at least one additional matrix dimensioned to cover the wound. In one embodiment, the first range of wavelengths ranges from 700 nanometers to 2000 nanometers, and the second range of wavelengths ranges from 380 nanometers to 700 nanometers. In one embodiment, the first range of wavelengths ranges from 700 nanometers to 900 nanometers, and the second range of wavelengths ranges from 380 nanometers to 410 nanometers.
In one embodiment, the kit further comprises a translucent film dimensioned to be positioned between the matrix and each light-emitting layer for connecting the matrix and at least one of the light-emitting layers. In one embodiment, the kit further comprises an antimicrobial adjuvant for synergistically potentiating antimicrobial activity of the light with respect to microbes on or adjacent the wound. In one embodiment, the antimicrobial adjuvant comprises a substituted naphthalene. In one embodiment, the antimicrobial adjuvant comprises an oxygen substituted naphthalene. In one embodiment, the antimicrobial adjuvant comprises 2-methyl-1,4-naphthoquinone. In one embodiment, the antimicrobial adjuvant is associated with the matrix. The matrix can include an associated bioactive agent selected from the group consisting of cells, drugs, precursors, enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, pharmaceuticals, photosensitizers, essential oils, therapeutics, and mixtures thereof.
In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of tetracyclines. In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of glycopeptide antibiotics. In one embodiment, the matrix has a surface including a channel, the surface facing the wound when the device is placed over the wound. In one embodiment, the kit includes a source of irrigation fluid that is configured to be placed in fluid communication with an inlet of the channel.
The range of wavelengths transmitted by the LEDs can range from 400-470 nm; from 600-700 nm, from 700-800 nm, from 800-1300 nm, comprising blue light, green light, red light, and near IR. The controller can be programmed to enable duty cycles ranging from 0% to 99% “on”; the matrix (e.g., bandage) layer would be relatively transparent enabling light transmission and either capable or not of being complexed with adjuvant antimicrobial substances. Wavelengths with or without additional antimicrobial substances can be tailored to attack either polymicrobial infections, gram-positive bacterial infections, gram-negative bacterial infections, anaerobic or aerobic infections, mycobacterial and fungal or mold infections.
In another aspect, the disclosure provides a kit for improving healing behavior of a biological wound. The kit comprises: a plurality of matrix components, each matrix component having a first section and a second section, the first section of one matrix component being dimensioned to matingly engage the second section of an adjacent matrix component to form all or a part of a matrix dimensioned to cover the wound; a light-emitting layer comprising a plurality of light sources, the light-emitting layer being configured to irradiate the wound or a region adjacent the wound with light at a range of wavelengths; and a controller configured to be placed in electrical communication with the light sources of the light-emitting layer, the controller being configured to execute a program stored in the controller to activate the light sources of each light-emitting layer to irradiate the wound or the region adjacent the wound when the matrix is placed over the wound.
In one embodiment, the first section of the one matrix component comprises a tab extending outward from a side of the one matrix component, and the second section of the adjacent matrix comprises a recess dimensioned to matingly engage the tab of the one matrix component.
In one embodiment, the range of wavelengths ranges from 380 nanometers to 700 nanometers. In one embodiment, the range of wavelengths ranges 380 nanometers to 410 nanometers.
In one embodiment, the kit further comprises a translucent film dimensioned to be positioned between the matrix and the light-emitting layer for connecting the matrix and the light-emitting layer. In one embodiment, the kit further comprises an antimicrobial adjuvant for synergistically potentiating antimicrobial activity of the light with respect to microbes on or adjacent the wound. In one embodiment, the antimicrobial adjuvant comprises a substituted naphthalene. In one embodiment, the antimicrobial adjuvant comprises an oxygen substituted naphthalene. In one embodiment, the antimicrobial adjuvant comprises 2-methyl-1,4-naphthoquinone. In one embodiment, the antimicrobial adjuvant is associated with the matrix. In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of cells, drugs, precursors, enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, pharmaceuticals, photosensitizers, essential oils, therapeutics, and mixtures thereof. In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of tetracyclines. In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of glycopeptide antibiotics. In one embodiment, the matrix has a surface including a channel, the surface facing the wound when the device is placed over the wound. In one embodiment, the kit further comprises a source of irrigation fluid configured to be placed in fluid communication with an inlet of the channel.
A combination of wavelengths (e.g. antimicrobial blue light with a wavelength of 400 nm, additional LEDs in the near IR wavelengths of 800-880 nm, or even 1280 nm can provide a combination of activities. 400 nm blue light can provide antimicrobial activity; 800-880 nm can enhance the killing of microbes in wound biofilm, acting synergistically with 400 nm. The 800-880 nm would additionally stimulate the infected host's healing response, further providing enhanced healing. Finally, higher wavelength such as 1280 nm can enhance immune activity of the infected host, further enhancing wound healing.
In another aspect, the disclosure provides a kit for improving healing behavior of a biological wound. The kit comprises: a matrix dimensioned to cover the wound; a light-emitting layer comprising a plurality of light sources, the light-emitting layer being configured to irradiate the wound or a region adjacent the wound with light at a range of wavelengths including near IR to stimulate microbes in a biofilm, rendering them more sensitive to microbicidal wavelengths; a controller configured to be placed in electrical communication with the light sources of the light-emitting layer, the controller being configured to execute a program stored in the controller to activate the light sources of each light-emitting layer to irradiate the wound or the region adjacent the wound when the matrix is placed over the wound; and an antimicrobial adjuvant for synergistically potentiating antimicrobial activity of the light with respect to microbes on or adjacent the wound. The invention provides synergism of near IR stimulation of microbes to enhance the destruction by microbicidal wavelengths.
In one embodiment, the antimicrobial adjuvant comprises a substituted naphthalene. In one embodiment, the antimicrobial adjuvant comprises an oxygen substituted naphthalene. In one embodiment, the antimicrobial adjuvant comprises 2-methyl-1,4-naphthoquinone. In one embodiment, the antimicrobial adjuvant is associated with the matrix. In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of cells, drugs, precursors, enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, pharmaceuticals, photosensitizers, essential oils, therapeutics, and mixtures thereof. In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of tetracyclines. In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of glycopeptide antibiotics.
In one embodiment, the matrix has a surface including a channel, the surface facing the wound when the device is placed over the wound. In one embodiment, the kit further comprises a source of irrigation fluid configured to be placed in fluid communication with an inlet of the channel.
In one embodiment, the range of wavelengths ranges from 380 nanometers to 700 nanometers. In one embodiment, the range of wavelengths ranges 380 nanometers to 410 nanometers.
In one embodiment, the kit further comprises a translucent film dimensioned to be positioned between the matrix and the light-emitting layer for connecting the matrix and the light-emitting layer.
Adjuvant antimicrobial substances can comprise vitamin K3 or menadione; tetracycline antibiotics which are activated by light; naturally occurring essential oils such as carvacrol; photo sensitizers such as methylene blue, with or without potassium iodide, EDTA can be included as an adjuvant. Additional photosensitizers such as chlorin E6 and ALA can be used.
In another aspect, the disclosure provides a method for improving healing behavior of a biological wound. The method comprises: (a) placing an antimicrobial adjuvant on or adjacent the wound; (b) covering at least a portion of the wound with a matrix; (c) irradiating the wound or a region adjacent the wound with light for a period of time, wherein the antimicrobial adjuvant synergistically potentiates antimicrobial activity of the light with respect to microbes on or adjacent the wound.
In one embodiment, the light has a wavelength that ranges from 380 nanometers to 500 nanometers. In one embodiment, the light has a wavelength that ranges from 380 nanometers to 700 nanometers. In one embodiment, the light is provided at a radiant exposure of at least 50 J/cm2 during the period of time.
In one embodiment, step (c) comprises: (i) irradiating the wound or the region adjacent the wound with light at a first range of wavelengths, and (ii) irradiating the wound or the region adjacent the wound with light at a second range of wavelengths, the first range of wavelengths being different than the second range of wavelengths.
In one embodiment, the first range of wavelengths ranges from 700 nanometers to 2000 nanometers, and the second range of wavelengths ranges from 380 nanometers to 700 nanometers. In one embodiment, irradiating the wound or the region adjacent the wound with light at the second range of wavelengths begins after irradiating the wound or the region adjacent the wound with light at the first range of wavelengths.
In one embodiment, the antimicrobial adjuvant comprises a substituted naphthalene. In one embodiment, the antimicrobial adjuvant comprises an oxygen substituted naphthalene. In one embodiment, the antimicrobial adjuvant comprises 2-methyl-1,4-naphthoquinone. In one embodiment, the antimicrobial adjuvant is associated with the matrix. In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of cells, drugs, precursors, enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, pharmaceuticals, photosensitizers, essential oils, therapeutics, and mixtures thereof. In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of tetracyclines. In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of glycopeptide antibiotics.
In one embodiment, the irrigation fluid comprises an additive. The additive can be selected from the group consisting of cells, drugs, precursors, enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, pharmaceuticals, photosensitizers, essential oils, therapeutics, and mixtures thereof. In one embodiment, the irrigation fluid comprises oxygen.
The 800-880 nm wavelengths can additionally stimulate the infected host's healing response, further providing enhanced healing. Finally, higher wavelengths such as 1280 nm can enhance immune activity of the infected host, further enhancing wound healing.
In another aspect, the disclosure provides a method for improving healing behavior of a biological wound. The method comprises: (a) covering at least a portion of the wound with a matrix having surface including a channel, the surface facing the wound when the matrix covers at least the portion of the wound; (b) irradiating the wound or a region adjacent the wound with light for a period of time; and (c) feeding an irrigation fluid to an inlet of the channel to irrigate the wound.
In one embodiment, the light has a wavelength that ranges from 380 nanometers to 500 nanometers. In one embodiment, the light has a wavelength that ranges from 380 nanometers to 700 nanometers. In one embodiment, the light is provided at a radiant exposure of at least 50 J/cm2 during the period of time.
In one embodiment, step (b) comprises: (i) irradiating the wound or the region adjacent the wound with light at a first range of wavelengths, and (ii) irradiating the wound or the region adjacent the wound with light at a second range of wavelengths, the first range of wavelengths being different than the second range of wavelengths. In one embodiment, step (a) comprises placing an antimicrobial adjuvant on or adjacent the wound, wherein the antimicrobial adjuvant synergistically potentiates antimicrobial activity of the light with respect to microbes on or adjacent the wound.
In one embodiment, the antimicrobial adjuvant comprises a substituted naphthalene. In one embodiment, the antimicrobial adjuvant comprises an oxygen substituted naphthalene. In one embodiment, the antimicrobial adjuvant comprises 2-methyl-1,4-naphthoquinone. In one embodiment, the antimicrobial adjuvant is associated with the matrix. In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of cells, drugs, precursors, enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, pharmaceuticals, photosensitizers, essential oils, therapeutics, and mixtures thereof. In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of tetracyclines. In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of glycopeptide antibiotics.
In one embodiment, the irrigation fluid comprises an additive. The additive can be selected from the group consisting of cells, drugs, precursors, enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, pharmaceuticals, photosensitizers, essential oils, therapeutics, and mixtures thereof. In one embodiment, the irrigation fluid comprises oxygen.
Adjuvant antimicrobial substances can comprise vitamin K3 or menadione; tetracycline antibiotics which are activated by light; naturally occurring essential oils such as carvacrol; photo sensitizers such as methylene blue, with or without potassium iodide, EDTA can be included as an adjuvant. Additional photosensitizers such as chlorin E6 and ALA can be used.
In another aspect, the disclosure provides a method for killing or inhibiting growth of microbes in a biofilm. The method comprises: (a) placing an antimicrobial adjuvant on or adjacent the biofilm; (b) irradiating the wound or a region adjacent the biofilm with light for a period of time, wherein the antimicrobial adjuvant synergistically potentiates antimicrobial activity of the light with respect to microbes on or adjacent the biofilm.
In one embodiment, the light has a wavelength that ranges from 380 nanometers to 700 nanometers. In one embodiment, step (b) comprises: (i) irradiating the biofilm or the region adjacent the biofilm with light at a first range of wavelengths, and (ii) irradiating the biofilm or the region adjacent the biofilm with light at a second range of wavelengths, the first range of wavelengths being different than the second range of wavelengths. In one embodiment, the first range of wavelengths ranges from 700 nanometers to 2000 nanometers, and the second range of wavelengths ranges from 380 nanometers to 700 nanometers. In one embodiment, irradiating the biofilm or the region adjacent the biofilm with light at the second range of wavelengths begins after irradiating the biofilm or the region adjacent the biofilm with light at the first range of wavelengths. In one embodiment, the light is provided at a radiant exposure of at least 50 J/cm2 during the period of time.
In one embodiment, step (a) comprises placing an antimicrobial adjuvant on or adjacent the biofilm, wherein the antimicrobial adjuvant synergistically potentiates antimicrobial activity of the light with respect to microbes on or adjacent the biofilm. In one embodiment, the antimicrobial adjuvant comprises a substituted naphthalene. In one embodiment, the antimicrobial adjuvant comprises an oxygen substituted naphthalene. In one embodiment, the antimicrobial adjuvant comprises 2-methyl-1,4-naphthoquinone. In one embodiment, step (a) comprises covering at least a portion of the biofilm with a matrix. In one embodiment, the antimicrobial adjuvant is associated with the matrix. The matrix can include an associated bioactive agent selected from the group consisting of cells, drugs, precursors, enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, pharmaceuticals, photosensitizers, essential oils, therapeutics, and mixtures thereof. In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of tetracyclines. In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of glycopeptide antibiotics.
In one embodiment, the irrigation fluid comprises an additive. The additive can be selected from the group consisting of cells, drugs, precursors, enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, pharmaceuticals, photosensitizers, essential oils, therapeutics, and mixtures thereof. In one embodiment, the irrigation fluid comprises oxygen.
In one embodiment, the microbes are bacteria, fungus, or mold on or adjacent a biological wound. In one embodiment, the bacteria are methicillin-resistant Staphylococcus aureus (MRSA). In one embodiment, the bacteria are Escherichia coli. In one embodiment, the bacteria are Pseudomonas aeruginosa.
Adjuvant antimicrobial substances can comprise vitamin K3 or menadione; tetracycline antibiotics which are activated by light; naturally occurring essential oils such as carvacrol; photo sensitizers such as methylene blue, with or without potassium iodide, EDTA can be included as an adjuvant. Additional photosensitizers such as chlorin E6 and ALA can be used.
Additionally, the combination of near infrared (e.g., 800-890 nm) stimulates microbial metabolic activity in biofilms, potentially enhancing the activity of cidal antibiotics, further aiding in antimicrobial activity.
In another aspect, the disclosure provides a method for killing or inhibiting growth of microbes in a biofilm. The method comprises: (a) covering at least a portion of the biofilm with a matrix having surface including a channel, the surface facing the biofilm when the matrix covers at least the portion of the biofilm; (b) irradiating the biofilm or a region adjacent the biofilm with light for a period of time; and (c) feeding an irrigation fluid to an inlet of the channel to irrigate the biofilm.
In one embodiment, the light has a wavelength that ranges from 380 nanometers to 700 nanometers. In one embodiment, step (b) comprises: (i) irradiating the biofilm or the region adjacent the biofilm with light at a first range of wavelengths, and (ii) irradiating the biofilm or the region adjacent the biofilm with light at a second range of wavelengths, the first range of wavelengths being different than the second range of wavelengths. In one embodiment, the first range of wavelengths ranges from 700 nanometers to 2000 nanometers, and the second range of wavelengths ranges from 380 nanometers to 700 nanometers. In one embodiment, irradiating the biofilm or the region adjacent the biofilm with light at the second range of wavelengths begins after irradiating the biofilm or the region adjacent the biofilm with light at the first range of wavelengths. In one embodiment, the light is provided at a radiant exposure of at least 50 J/cm2 during the period of time.
In one embodiment, step (a) comprises placing an antimicrobial adjuvant on or adjacent the biofilm, wherein the antimicrobial adjuvant synergistically potentiates antimicrobial activity of the light with respect to microbes on or adjacent the biofilm. In one embodiment, the antimicrobial adjuvant comprises a substituted naphthalene. In one embodiment, the antimicrobial adjuvant comprises an oxygen substituted naphthalene. In one embodiment, the antimicrobial adjuvant comprises 2-methyl-1,4-naphthoquinone. In one embodiment, step (a) comprises covering at least a portion of the biofilm with a matrix. In one embodiment, the antimicrobial adjuvant is associated with the matrix. The matrix can include an associated bioactive agent selected from the group consisting of cells, drugs, precursors, enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, pharmaceuticals, photosensitizers, essential oils, therapeutics, and mixtures thereof. In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of tetracyclines. In one embodiment, the matrix includes an associated bioactive agent selected from the group consisting of glycopeptide antibiotics.
In one embodiment, the irrigation fluid comprises an additive. The additive can be selected from the group consisting of cells, drugs, precursors, enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, pharmaceuticals, photosensitizers, essential oils, therapeutics, and mixtures thereof. In one embodiment, the irrigation fluid comprises oxygen.
In one embodiment, the microbes are bacteria, fungus, or mold on or adjacent a biological wound. In one embodiment, the bacteria are methicillin-resistant Staphylococcus aureus (MRSA). In one embodiment, the bacteria are Escherichia coli. In one embodiment, the bacteria are Pseudomonas aeruginosa.
Adjuvant antimicrobial substances can comprise vitamin K3 or menadione; tetracycline antibiotics which are activated by light; naturally occurring essential oils such as carvacrol; photo sensitizers such as methylene blue, with or without potassium iodide, EDTA can be included as an adjuvant. Additional photosensitizers such as chlorin E6 and ALA can be used.
Additionally, the combination of near infrared (e.g., 800-890 nm) stimulates microbial metabolic activity in biofilms, potentially enhancing the activity of cidal antibiotics, further aiding in antimicrobial activity.
It is an advantage of the invention to provide devices for improving healing behavior of a biological wound. The ability to use an LED layer with either 800-880 nm, or 1280 nm, or both would improve host wound healing of the wound. The lower wavelength enhances tissue regrowth and healing, while the higher wavelength stimulates immune cell function, both serving to improve wound healing.
It is another advantage of the invention to provide methods for improving healing behavior of a biological wound.
It is another advantage of the invention to provide kits for improving healing behavior of a biological wound. The ability to use an LED layer with either 800-880 nm, or 1280 nm, or both would improve host wound healing of the wound. The lower wavelength enhances tissue regrowth and healing, while the higher wavelength stimulates immune cell function, both serving to improve wound healing.
It is another advantage of the invention to provide methods for killing or inhibiting growth of microbes in a biofilm such as bacteria in a biofilm on or adjacent a biological wound.
It is another advantage of the invention to provide a device for combining both antimicrobial light therapy and light-enhanced wound and inflammation healing.
These and other features, aspects and advantages of various embodiments of the present invention will become better understood with regard to the following description, appended claims, and accompanying Figures.
Like reference numerals will be used to refer to like parts from Figure to Figure in the following detailed description. Any drawings herein are not shown to scale. Where dimensions are given in the text or figures, these dimensions are merely example values which could be used with one or more example implementations and do not limit the scope of the disclosed invention.
The present disclosure provides methods and devices for improving healing behavior of a biological wound, kits for improving healing behavior of a biological wound, and methods for killing or inhibiting growth of microbes in a biofilm such as bacteria in a biofilm on or adjacent a biological wound.
Before the present invention is described in further detail, it is to be understood that the invention is not limited to the particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The scope of the present invention will be limited only by the claims. As used herein, the singular forms “a”, “an”, and “the” include plural embodiments unless the context clearly dictates otherwise.
It should be apparent to those skilled in the art that many additional modifications beside those already described are possible without departing from the inventive concepts. In interpreting this disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. Variations of the term “comprising”, “including”, or “having” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, so the referenced elements, components, or steps may be combined with other elements, components, or steps that are not expressly referenced. Embodiments referenced as “comprising”, “including”, or “having” certain elements are also contemplated as “consisting essentially of” and “consisting of” those elements, unless the context clearly dictates otherwise. It should be appreciated that aspects of the disclosure that are described with respect to a system are applicable to the methods, and vice versa, unless the context explicitly dictates otherwise.
Furthermore, relative terms, such as “lower” or “bottom,” “upper” or “top,” “left” or “right,” “above” or “below,” “front” or “rear,” may be used herein to describe one element's relationship to another element as illustrated in the Figures. It will be understood that relative terms are intended to encompass different orientations of the device in addition to the orientation depicted in the Figures.
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Various configurations of the light-emitting diodes are possible. In the sub-array at the lower left corner of the light emitting layer 220, eight light-emitting diodes 224 surround a single light-emitting diode 226. The light-emitting diodes 224 emit light having a first range of wavelengths ranging from 700 nanometers to 2000 nanometers, or 700 nanometers to 1000 nanometers, or 700 nanometers to 900 nanometers. The light-emitting diode 226 emits light having a second range of wavelengths ranging from 380 nanometers to 700 nanometers, or 380 nanometers to 420 nanometers, or 380 nanometers to 410 nanometers. The sub-array pattern of eight light-emitting diodes 224 surrounding a single light-emitting diode 226 can be over the entire surface 222 of the light emitting layer 220.
In the sub-array at the lower right corner of the light emitting layer 220, alternating columns of light-emitting diodes 224 and light-emitting diodes 226 are used. The light-emitting diodes 224 emit light having a first range of wavelengths ranging from 700 nanometers to 2000 nanometers, or 700 nanometers to 1000 nanometers, or 700 nanometers to 900 nanometers. The light-emitting diodes 226 emit light having a second range of wavelengths ranging from 380 nanometers to 700 nanometers, or 380 nanometers to 420 nanometers, or 380 nanometers to 410 nanometers. The sub-array pattern of alternating columns of light-emitting diodes 224 and light-emitting diodes 226 can be over the entire surface 222 of the light emitting layer 220.
In the sub-array at the upper right corner of the light emitting layer 220, the light-emitting diodes 224 and light-emitting diodes 226 alternate in each row and column. The light-emitting diodes 224 emit light having a first range of wavelengths ranging from 700 nanometers to 2000 nanometers, or 700 nanometers to 1000 nanometers, or 700 nanometers to 900 nanometers. The light-emitting diodes 226 emit light having a second range of wavelengths ranging from 380 nanometers to 700 nanometers, or 380 nanometers to 420 nanometers, or 380 nanometers to 410 nanometers. The sub-array pattern of alternating in the rows and columns of light-emitting diodes 224 and light-emitting diodes 226 can be over the entire surface 222 of the light emitting layer 220.
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Any of the matrices 110, 210, 210A, 310, and 410 or the matrix component 210B of the devices 110, 200, 300, and 400 may comprise a biocompatible material such as polyethylene, polypropylene, nylon, polyester, polyurethane, polyvinyl chloride, polyvinyl alcohol, polyvinylidene chloride, polyethylene/vinyl acetate copolymer, polyethylene/acrylic acid copolymer, polydimethylsiloxane (PDMS), and the like. The biocompatible material may be drug-eluting. Any of the matrices 110, 210, 210A, 310, and 410 of the devices 110, 200, 300, and 400 may have a thickness in a range of 0.5 millimeters to 20 millimeters, or 0.5 millimeters to 10 millimeters, or 2 millimeters to 8 millimeters.
Any of the devices 100, 200, 300, or 400 may be used in one non-limiting example method according to the invention for improving healing behavior of a biological wound in a body part of a subject. The “subject” can be a mammal, preferably a human. In the method, an antimicrobial adjuvant is placed on or adjacent the wound 102, and at least a portion of the wound 102 is covered with a matrix (e.g., any of the matrices 110, 210, 210A, 310, and 410). Various methods can be used to place the antimicrobial adjuvant on or adjacent the wound 102. For example, the antimicrobial adjuvant may be directly applied on or adjacent the wound 102, and then at least a portion of the wound 102 is covered with a matrix of any of the devices 100, 200, 300, or 400. Alternatively, an antimicrobial adjuvant is “associated” with the matrix by being directly or indirectly, physically or chemically bound to the matrix. Non-limiting examples of chemical bonds include covalent bonds, ionic bonds, coordinate bonds, and hydrogen bonds. Indirect bonding can include the use of a group of atoms (i.e., a linker) that chemically links the bioactive agent and the matrix. Non-limiting examples of physical bonding include physical adsorption, absorption, and entrapment. The antimicrobial adjuvant can elute from an interior of the matrix to a location on or adjacent the wound, or from a surface of the matrix that faces the wound when the matrix covers the wound. The wound or a region adjacent the wound is irradiated with light from the light-emitting layer (e.g., any of the light-emitting layers 120, 220, 320, and 420 for a period of time, preferably for a period of time such that the light is provided at a radiant exposure of at least 50 J/cm2 during the period of time. The antimicrobial adjuvant synergistically potentiates antimicrobial activity of the light with respect to microbes on or adjacent the wound. In one embodiment, the method does not require additional antimicrobial agents. In one embodiment, the antimicrobial adjuvant comprises a substituted naphthalene. In one embodiment, the antimicrobial adjuvant comprises an oxygen substituted naphthalene. In one embodiment, the antimicrobial adjuvant comprises 2-methyl-1,4-naphthoquinone (see
Any of the devices 100, 200, 300, or 400 may be used in another non-limiting example method according to the invention for improving healing behavior of a biological wound in a body part of a subject. In the method, the plurality of light sources comprises an array of light emitting diodes in electrical communication with the controller, and the controller is configured to execute a program stored in the controller to: (i) activate a first group of the light emitting diodes to irradiate the wound or the region adjacent the wound with light at a first range of wavelengths, and (ii) thereafter activate a second group of the light emitting diodes to irradiate the wound or the region adjacent the wound with light at a second range of wavelengths. The first range of wavelengths (e.g., 700-900 nanometers) is different than the second range of wavelengths (e.g., 380-410 nanometers), and the light at the first range of wavelengths functions as an antimicrobial adjuvant for the light at a second range of wavelengths.
Any of the devices 100, 200, 300, or 400 may be used in another non-limiting example method according to the invention for improving healing behavior of a biological wound in a body part of a subject. In the method, at least a portion of the wound 102 is covered with a matrix (e.g., any of the matrices 110, 210, 210A, 310, and 410), and the wound or a region adjacent the wound is irradiated with light from the light-emitting layer (e.g., any of the light-emitting layers 120, 220, 320, and 420 for a period of time, preferably for a period of time such that the light is provided at a radiant exposure of at least 50 J/cm2 during the period of time. An irrigation fluid is fed to an inlet of one or more channels (e.g., any of channels 112, 212, 212A, or 412) of the matrix to irrigate the wound. An irrigation fluid is supplied via a conduit (e.g., 474 of device 400) and enters an inlet of a channel (e.g., 412 of device 400), and passes over the wound 102, and exits an outlet of the channel via a conduit (e.g., 475 of device 400) that acts as a waste collector for the used irrigation fluid. The irrigation fluid may comprise an additive in a carrier such as water. The additive can be selected from the group consisting of cells, drugs, precursors, enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, pharmaceuticals, photosensitizers, essential oils, therapeutics, and mixtures thereof. Alternatively, the irrigation fluid can comprise oxygen. Also, an issue using phototherapy is potential overheating on the skin caused by long periods of light exposure and power consumption for a portable device. In order to solve this problem, the irrigation fluid may be provided at a suitable temperature (e.g., 20-25° C.) to act as a coolant for the wound and adjacent skin.
Any of the devices 100, 200, 300, or 400 may be used in another non-limiting example method according to the invention for improving healing behavior of a biological wound in a body part of a subject in which an antimicrobial adjuvant, light irradiation, and a bioactive agent are used. Any of the matrices 110, 210, 210A, 310, and 410 or the matrix component 210B of the devices 110, 200, 300, and 400 may be associated with a bioactive agent. A bioactive agent is “associated” with the matrix if the bioactive agent is directly or indirectly, physically or chemically bound to the matrix. Non-limiting examples of chemical bonds include covalent bonds, ionic bonds, coordinate bonds, and hydrogen bonds. Indirect bonding can include the use of a group of atoms (i.e., a linker) that chemically links the bioactive agent and the matrix. Non-limiting examples of physical bonding include physical adsorption, absorption, and entrapment. The bioactive agent can elute from an interior of the matrix to a location on or adjacent the wound, or from a surface of the matrix that faces the wound when the matrix covers the wound.
A “bioactive agent” as used herein includes, without limitation, physiologically or pharmacologically active substances that act locally or systemically in the body. A bioactive agent is a substance used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness, or a substance which affects the structure or function of the body or which becomes biologically active or more active after it has been placed in a predetermined physiological environment. Bioactive agents include, without limitation, cells, drugs, precursors, enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, pharmaceuticals, photosensitizers, essential oils, and therapeutics.
In one example embodiment, the matrix includes an associated bioactive agent selected from the group consisting of tetracyclines. In one example embodiment, the matrix includes an associated bioactive agent selected from minocycline, doxycycline, demeclocycline, and mixtures thereof. In one example embodiment, the matrix includes an associated bioactive agent selected from the group consisting of glycopeptide antibiotics, such as vancomycin.
Any of the devices 100, 200, 300, or 400 may be used in one non-limiting example method according to the invention for killing or inhibiting growth of microbes in a biofilm. Looking at
Any of the devices 100, 200, 300, or 400 may be used in another non-limiting example method according to the invention for killing or inhibiting growth of microbes in a biofilm. In the method, the plurality of light sources comprises an array of light emitting diodes in electrical communication with the controller, and the controller is configured to execute a program stored in the controller to: (i) activate a first group of the light emitting diodes to irradiate the biofilm or the region adjacent the biofilm with light at a first range of wavelengths, and (ii) thereafter activate a second group of the light emitting diodes to irradiate the biofilm or the region adjacent the biofilm with light at a second range of wavelengths. The first range of wavelengths (e.g., 700-900 nanometers) is different than the second range of wavelengths (e.g., 380-410 nanometers), and the light at the first range of wavelengths functions as an antimicrobial adjuvant for the light at a second range of wavelengths.
Any of the devices 100, 200, 300, or 400 may be used in another non-limiting example method according to the invention for killing or inhibiting growth of microbes in a biofilm. In the method, at least a portion of the biofilm 102 is covered with a matrix (e.g., any of the matrices 110, 210, 210A, 310, and 410), and the biofilm or a region adjacent the biofilm is irradiated with light from the light-emitting layer (e.g., any of the light-emitting layers 120, 220, 320, and 420 for a period of time, preferably for a period of time such that the light is provided at a radiant exposure of at least 50 J/cm2 during the period of time. An irrigation fluid is fed to an inlet of one or more channels (e.g., any of channels 112, 212, 212A, or 412) of the matrix to irrigate the biofilm. An irrigation fluid is supplied via a conduit (e.g., 474 of device 400) and enters an inlet of a channel (e.g., 412 of device 400), and passes over the biofilm, and exits an outlet of the channel via a conduit (e.g., 475 of device 400) that acts as a waste collector for the used irrigation fluid. The irrigation fluid may comprise an additive in a carrier such as water. The additive can be selected from the group consisting of cells, drugs, precursors, enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, pharmaceuticals, photosensitizers, essential oils, therapeutics, and mixtures thereof. Alternatively, the irrigation fluid can comprise oxygen.
Embodiments of the invention, such as the non-limiting example embodiments of the devices 100, 200, 300, or 400, may use a flexible optical fiber dressing system with a blue light source, and may keep colony counts below 105/gram of tissue, thereby reducing bacterial bioburden. Beneficial features of some embodiments of the invention may include: Optional features for particular embodiments may include: not harmful to host tissue (able to “breath”); able to handle exudate without “fouling”; broad antimicrobial effect (gram positive and gram negative pathogens); highly transparent; flexible and capable of being placed into irregular spaces; inexpensive components, simple manufacture; FDA approved materials; light weight; and battery-operated. Device considerations include: power density and heat dissipation. For the biology of infection, the device is intended to prevent and treat wound contamination and infection, and reduce bacterial bioburden and wound infections rapidly becoming biofilm.
The following Examples are provided to demonstrate and further illustrate certain embodiments and aspects of the present invention and are not to be construed as limiting the scope of the invention.
In this Example 1, we show that Vitamin K3 (menadione) acts as a photosensitizer to synergize with blue light to kill drug-resistant bacteria in biofilms.
Antimicrobial resistance (AMR) is a threat to global human health of paramount significance, threatening to once again return common bacterial infections to major causes of death [Ref. 2-4]. It is universally acknowledged that the prolonged use, and overuse, of antibiotics in many settings is a major contributor [Ref. 2-4].
In a study of thousands of wound infections, in which antibiotics were antibiotic-susceptibility matched, treatment-induced resistance often resulted from rapid reinfection with a different strain, resistant to that antibiotic. The more prolonged and chronic the treatment, the more likely the development of that resistance. Complicating wound treatment is the fact that wound infections rapidly develop into biofilm infections, in which bacteria are encased in a matrix or glycocalyx of bacterial carbohydrates, bacterial and host DNA, bacterial and host proteins, all further rendering biofilm-resident bacteria dramatically less sensitive to antibiotics [Ref. 26] In fact, bacteria in biofilms are 100-1000 times less sensitive to antibiotics [Ref. 27]. Thus, alternatives to antibiotics, or ways of augmenting their activity, are becoming a necessity. Unfortunately, phototherapy is often conspicuously omitted as a technology to reduce bacterial infection while avoiding the development of antibiotic resistance [Ref. 4].
Many wound infections, especially cutaneous infections, are accessible to light. Light as a therapy against microbial infection was discovered over 100 years ago. More recently, blue light, in wavelengths from 400-470 nm, have been found to be effective in experimental biofilm infections both in vitro and in vivo [Ref. 6, 27]. It is now well-established that blue light, particularly at a wavelength of 405 nm, has potent in vitro and in vivo antimicrobial activities. These have been extensively reviewed [Ref. 6, 14]. At least one mechanism by which antimicrobial blue light (aBL) functions is through its ability to interact with endogenous chromophores within bacterial cells, especially intracellular porphyrins and flavins in the microbial cells. Though not fully understood, evidence supports the hypothesis that the absorption of photon energy of blue light by the bacterial chromophore results in the generation of reactive oxygen species, most notably singlet oxygen, superoxide, and hydrogen peroxide intracellularly in the bacteria, which do not have the same efficient antioxidant capabilities of mammalian cells. Depending on the bacterial species, microbial culture conditions, and the many variables of light administration (light/power density, total dose, environmental temperature, etc.), bactericidal activity in the realm of two-seven logs of killing may be achievable in vitro in planktonic cultures [Ref. 27]. Similar activities have been described in small animal models of infection, but no systematic studies have been carried out using antimicrobial blue light in large animals [Ref. 6].
A recent paper [Ref. 28] examining the effectiveness of antimicrobial therapy for diabetic foot infections found that 95% of the predominant pathogens were gram-positive cocci, the most virulent and resistant of which is methicillin-resistant Staphylococcus aureus, or MRSA. Cutaneous wound infections, including diabetic foot ulcers, are exceedingly common, often chronic, and commonly associated with biofilms that both protect the bacteria and further slow wound healing [Ref. 6]. Due to the very nature of these wounds, the combination of bacterial contamination and chronicity of infection makes prolonged antibiotic therapy currently inevitable. A landmark 2014 study in the U.S. [Ref. 3] involving over 32,000 matched pairs of diabetic patients, either with or without diabetic foot ulcers (DFU), found that the cost of DFU's alone contributed $9-$13 billion annually to the cost of diabetic care, and resulted in 4-5% of patients requiring amputation.
In this Example 1, we studied the effectiveness of aBL against MRSA, as well as other pathogens, in vitro and in an ex vivo porcine skin wound model (the closest animal simulant to human skin). The latter was chosen for preclinical screening [Ref. 29,30] before beginning large animal testing of blue light treatments for cutaneous wounds in vivo in swine, a necessary regulatory prelude to clinical trials. In the course of developing our ex vivo porcine skin model [Ref. 30] for evaluating phototherapy, we noted a substantial decrease in the bactericidal activity of aBL when tested in vitro and ex vivo in biofilms as compared with planktonic cultures.
Brynildsen et al. [Ref. 31] had previously described the use of menadione, or vitamin K3, to augment bacterial intracellular ROS production. Menadione was reported to have multiple antimicrobial activities and was suggested as a potential topical agent [Ref. 9]. Menadione has been FDA approved for oral administration and is still widely included in animal feed as a dietary supplement. Menadione is a quinone, an analog of 1,4-naphthoquinone (see
Stock solutions of menadione (10 mg/mL) were prepared in DMSO only and in DMSO/Tween (1:1) to improve the solubility. Different concentrations of menadione were prepared to make a calibration curve by diluting the stock solution in PBS so that the final solution was either 1% DMSO or 1% DMSO/Tween. The absorbance spectra were measured using an Evolution™ 300 UV-Vis Spectrophotometer (Thermo Scientific, Waltham, Massachusetts, USA). The calibration curve was measured, and molar absorption coefficient calculated.
Menadione (Mn), Dimethyl sulfoxide (DMSO), and Tween 80 were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Phosphate-buffered saline (PBS) for microbial cell suspension and serial dilution and Brain-heart infusion broth (BHI) were purchased from Fisher Scientific (Waltham, Massachusetts, USA). Menadione stock solution of 100 mg/mL was prepared by dissolving it in a mixture of DMSO and Tween 80 in the ratio of 1:1 and was diluted to the final concentration in PBS before use.
As a light source for aBL irradiation, a light-emitting diode (LED; M405L4; Thorlabs, USA) with a peak emission of 405 nm and a full width at half maximum of 25 nm was used. The power density/irradiance (mW/cm2) of transmitted light energy was established by measuring the surface of the target with the use of a PM100D power meter (Thorlabs, USA). We used different power densities: 50, 30, 15 and 10 mW/cm2. The radiant exposure (also referred to as fluence or dose) was calculated with the following equation (Radiant exposure (J/cm2)=Irradiance (W/cm2)×Exposure time(s)).
The strains used in this study were methicillin-resistant Staphylococcus aureus (USA300 Lux, AF0003, IQ0064), Pseudomonas aeruginosa PA01 (P. aeruginosa) and Escherichia coli ATCC 25922 (E. coli). These strains were routinely cultured on BHI agar plates at 37° C. in 5% CO2.
The MICs of menadione in different bacterial strains were determined by a standard broth microdilution assay according to the Clinical and Laboratory Standards Institute. Briefly, menadione was prepared at 100 mg/mL in Tween 80/DMSO (1:1) as a stock solution. The stock solution was diluted to 2048 μg/mL in BHI and then serial dilutions were performed to 1024-0.015 μg/mL in a 96-well plate. Then, a 10 μL stationary growth-phase culture of 108 CFU/mL bacteria in BHI broth was added to the 96-well plate containing the serial dilutions of menadione. The medium containing a similar amount of Tween 80/DMSO (1:1) served as a control. The microplates were incubated at 37° C. for 24 hours and the lowest concentration of the compound capable of completely inhibiting bacterial growth was referred to as the MIC.
The bacterial cells were grown in BHI broth medium shaken overnight. Cells were collected by centrifugation at 4,000 rpm for 5 minutes and suspended in PBS at a density of 108 colony-forming units (CFU)/mL (estimated by measuring the optical density at 600 nm; O.D 0.1=108 cells/mL). The microbial suspension in PBS was transferred to a 35×12-mm dish and mixed with different concentrations of menadione: 0, 0.5, 1 and 2 μg/mL for MRSA USA300, AF003 and IQ0064; 0, 1, 10 and 30 μg/mL for P. aeruginosa PA01; and 0, 16, 32, 64 and 128 μg/mL for E. coli ATCC 25922. After incubation in the dark at 25° C. for 1 hour, the planktonic suspension of cells at room temperature was irradiated using aBL (405 nm) at 50 mW/cm2 with different radiant exposures (0-250 J/cm2). During the aBL irradiation, 40 μL of samples were withdrawn every 15 minutes, and the CFUs were determined by 10-fold serial dilutions in PBS (10-1 to 10-7 dilution factors), plated on BHI agar plates as described previously [Ref. 17]. CFUs were counted after overnight incubation at 37° C. Experiments were performed in triplicate.
The bacterial suspension was grown in a shaking incubator overnight in BHI broth. Cells were collected by centrifugation at 4,000 rpm for 5 minutes and suspended in PBS at a density of 106 CFU/mL. Then, the biofilms were grown for 48 hours (unless otherwise specified) in 96-well microtiter plates as described previously [Ref. 27], with a renewal of the media every 24 hours. Following the 48-hour biofilm growth, fresh 200 μL of PBS with or without menadione in different concentrations were added to the wells and the biofilms were incubated in the dark at 25° C. for 1 hour: 0, 0.5, 1, and 2 μg/mL for MRSA USA300, AF0001, IQ0064; 0, 1, 10, 30 and 50 μg/mL for P. aeruginosa PA01; and 0, 100, and 150 μg/mL for E. coli ATCC 25922. After that, the control and the menadione-treated biofilms were maintained without irradiation (“menadione/dark”). Simultaneously, the aBL was delivered to the other biofilm groups testing different power densities (50, 30, 15, and 10 mW/cm2) while keeping the same radiant exposure of 250 J/cm2. After the light exposure, the biofilms were carefully washed two times with PBS and 200 μL of PBS was added to each well. Then, the bacterial biofilms in each well were harvested by scratching with a sterile pipette tip and three wells of each group were pooled together in a 1.5 mL microcentrifuge tube. The total volume obtained, 600 μL (3 wells), was sonicated using a Branson 2510 Water Bath Sonicator (Marshall Scientific, LLC) for 5 minutes and the CFU was determined by 10-fold serial dilutions in PBS on BHI agar plates. CFU was counted after overnight incubation at 37° C. Experiments were performed in triplicate.
Skin was handled and sanitized with 70% isopropanol solution after hair shaving. The explants and inner wounds were created using a 12 mm and 4 mm biopsy punches respectively. The explants were placed in 12 wells plates using a (8 μm pore) inserts (Corning™ Falcon™). 1 mL of DMEM media was added to each well in the plate before the insert. After 24 hours, the inner wounds in the explants were inoculated with 20 μL of 108 CFU/mL of bacterial solution. After that, the explants were incubated at 37° C. and 5% CO2 for 48 hours to ensure the growth of a mature bacterial biofilm. The inner wounds of the explants were replenished daily with fresh BHI media to ensure that the wounds are hydrated.
1.2.9.1 General ROS measured by 2′,7′-dichlorofluorescein (DCFH-DA) assay: DCF-DA was purchased from Thermofisher. This probe is chemically reduced and acetylated in nonfluorescent form until the acetate groups are removed by intracellular esterase and oxidation [Ref. 22]. The production of ROS by menadione when combined with blue light was measured in the presence and absence of bacteria. The bacteria (MRSA) was grown in BHI broth medium with shaking overnight. Cells were collected by centrifugation at 4,000 rpm for 5 minutes and resuspended in PBS in the stationary growth phase culture of 1×108 CFU/mL. The microbial suspension was transferred to a 35×12-mm dish, mixed with 10 μg/mL of menadione, and stained with 10 μM DCFH-DA solution per the manufacturer's instruction. The solutions (with and without bacteria) were irradiated with the blue light at 50 mW/cm2 and the samples were collected every 15 minutes to be analyzed by Microplate Spectrofluorometer (SPECTRAmax®, Molecular Devices, USA) at λex=495 nm and λem=525 nm. The control and the menadione/dark groups were generated under the same conditions without irradiation.
1.2.9.2 Singlet oxygen 1O2 assay: The singlet oxygen was measured by Singlet Oxygen Sensor Green Reagent (SOSG) (Thermofisher). In the presence of singlet oxygen, SOSG emits a green fluorescence similar to that of fluorescein (excitation/emission maxima ˜504/525 nm). The MRSA were grown in BHI broth medium with shaking overnight. Cells were collected by centrifugation at 4,000 rpm for 5 minutes and resuspended in PBS in the stationary growth phase culture of 1×108 CFU/mL. The microbial suspension was transferred to a 35×12-mm dish, mixed with 10 μg/mL of menadione, and stained with 10 μM SOSG solution per the manufacturer's instruction. The bacteria were irradiated with the blue light at 50 mW/cm2 and the samples were collected each 15 minutes to be analyzed by Microplate Spectrofluorometer (SPECTRAmax®, Molecular Devices, USA) at λex=504 nm and λem=525 nm. The control and the menadione/dark groups were generated under the same conditions without irradiation.
1.2.9.3 Hydroxyl radical HO′ assay: The hydroxyl radical was measured by 3′-(p-hydroxyphenyl) fluorescein (HPF) (Thermofisher). HPF is a new fluorescein derivative, and it is nonfluorescent until reacted with the hydroxyl radical or peroxynitrite anion. The MRSA were grown in BHI broth medium with shaking overnight. Cells were collected by centrifugation at 4,000 rpm for 5 minutes and resuspended in PBS in the stationary growth phase culture of 1×108 CFU/mL. The microbial suspension was transferred to a 35×12-mm dish, mixed with 10 μg/mL of menadione, and stained with 10 μM HPF solution per the manufacturer's instruction. The bacteria were irradiated with the blue light at 50 mW/cm2 and the samples were collected each 15 minutes to be analyzed by Microplate Spectrofluorometer (SPECTRAmax®, Molecular Devices, USA) at λex=490 nm and λem=515 nm. The control and the menadione/dark groups were generated under the same conditions without irradiation.
The chemical structure of the menadione is shown in
Vancomycin is a common antibiotic that is used to treat serious MRSA skin infectious. We tested different concentrations of vancomycin (5-500 μg/mL) combined with blue light (50 mW/cm2-250 J/cm2) to treat MRSA 48 hours biofilm. Even the higher concentration of vancomycin, 500 μg/mL, which is 25 times above normally safe achievable serum levels, increased killing only by 2.65 log10 CFU/mL in the MRSA biofilm compared to 1.93 log10 CFU/mL of aBL alone. The lower concentrations of vancomycin 5, 15 and 50 μg/mL did not show any improvement of aBL antimicrobial effect (
Menadione demonstrated antibacterial activity with an MIC of 8 μg/mL, 512 μg/mL, and 1024 μg/mL against MRSA, E. coli, and P. aeruginosa respectively in the absence of blue light or any ambient light. However, we investigated the effects of aBL (50 mW/cm2) in potentiating the antibacterial activities of menadione in the three strains of bacteria. Different fluence values were investigated in different ranges (125 J/cm2-15.62 J/cm2). aBL effectively reduced the MIC values observed when added to menadione. Starting with the lowest fluence value investigated (15.62 J/cm2), the menadione MIC values were found to be 8 μg/mL, 256 μg/mL, and 64 μg/mL for MRSA, E. coli, and P. aeruginosa respectively. It is important to note that aBL is highly effective as an antibacterial since it was able to reduce the MIC of E. coli and P. aeruginosa in as little as 5 minutes irradiating interval. However, the MIC of menadione for MRSA did not change at this fluence rate. Additionally, irradiating the bacteria for 10.5 minutes (31.25 J/cm2) had decreased the menadione MIC values of all bacterial strains to the following values: 4 μg/mL, 256 μg/mL, and 8 μg/mL for MRSA, E. coli, and P. aeruginosa respectively. Furthermore, the fluence of 62.5 J/cm2 achieved complete eradication of P. aeruginosa and lowered the MIC values for MRSA and E. coli to 1 μg/mL and 4 μg/ml respectively. This suggests that at even low concentrations, menadione can enhance the microbicidal effectiveness of blue light. The highest fluence that we investigated was 125 J/cm2, when we irradiated the bacteria for 42 minutes. Irradiating for this duration at this power density with aBL has completely eradicated all E. coli and P. aeruginosa and lowered the MIC of menadione for MRSA for as low as 0.0625 μg/mL which is 78% lower. Therefore, we observed that the treatment combination of menadione inoculation and aBL irradiation resulted in significantly lower menadione MIC values.
E. coli
P. aeruginosa
Menadione potentiated the effect of aBL in the planktonic cultures of MRSA, P. aeruginosa, and E. coli.
After 48 hours of incubation, mature monomicrobial biofilms of MRSA, P. aeruginosa, and E. coli were obtained with 8.00 log10 CFU/mL per well. After the treatment with the aBL alone at 250 J/cm2 (50 mW/cm2), the log10 photoinactivation for each species was 2.14, 4.45, and 3.83 log10 CFU/mL for MRSA, P. aeruginosa and E. coli, respectively. When the biofilms were treated with menadione and 250 J/cm2 (50 mW/cm2) aBL combined, all the strains showed increased eradication. In
Temperature was recorded during the aBL irradiation (50 m W/cm2-250 J/cm2-83 minutes) in the 96-well microtiter plates through a thermocouple (OMEGA RDXL4SD). The final temperature measured was 29.9° C., with an increase of only 5° C. from the initial temperature, which did not affect the viability of the microorganisms. Despite the fact that this is below the threshold for pain in human skin, considering the possible intolerance of human tissue to even mild heat, the monomicrobial biofilms of MRSA were also treated with menadione and aBL in lower power densities as 30, 15, and 10 mW/cm2. The enhancement of the antimicrobial effect of aBL with menadione was observed even with low power density. At 15 mW/cm2 and 250 J/cm2 of aBL, 3.01 log10 CFU/mL of MRSA biofilm were killed in the presence of 1 μg/mL of Menadione while the aBL alone in the same conditions was able to inactivate only 1.48 log10 CFU/mL, approximately 102 less (see Table 3).
48 hours following the inoculation, mature monobacterial biofilms of MRSA, P. aeruginosa, and E. coli were obtained with 8.00 log10 CFU/g per wound bed on each explant. MRSA biofilms in explant were reduced by 0.81 log10 CFU/g after the treatment of just aBL 250 J/cm2 (50 mW/cm2). However, MRSA biofilms in ex-vivo porcine skin were reduced by up to 4.18 log10 CFU/g when treated with aBL combined with menadione (200 μg/mL). For MRSA biofilms, the log reduction of mature monobacterial biofilm increased as the menadione concentration increased (see
Observing that there was some, albeit minimal, absorption of 405 nm light by menadione, and that together they synergistically increased bactericidal activity, we investigated possibility that menadione might be acting as a photosensitizer. To do so, the production of ROS by menadione combined with blue light was measured by different ROS probes, DCF-DA, SOSG and HPF, in the presence and absence of MRSA. The acetylated forms of 2′,7′-dichlorofluorescein (DCF-DA) is nonfluorescent until the acetate groups are removed by intracellular esterase and oxidation occurs within the cell. Oxidation of these probes can be detected by monitoring the increasing of fluorescence signals. The SOSG reagent is highly selective for 102 while the HPF is selective for OH. Both do not fluoresce until they react with their specific substrate, which means the signal of fluorescence is be related to specific ROS production. In
The general ROS production also was tested in presence or absence of MRSA to evaluate whether menadione was directing with the bacteria to potentiate the aBL effect or not. The result presented in
Our studies demonstrate the efficacy of Vitamin K3, menadione, to synergistically potentiate the antimicrobial effect of 405 nm blue light against several critical human multi-drug resistant (MDR) pathogenic bacteria. Antimicrobial resistance (AMR) has become a major public health threat worldwide [Ref. 4]. Despite current efforts, treatment options for MDR bacteria remain limited, and are only becoming more so as bacteria become increasingly resistant. The effect of aBL and its potential for multi-log killing of bacteria in bacterial planktonic cultures in different bacterial species, as well as many fungi, is well documented in the literature [Ref. 8].
It is interesting to note that in the recent comprehensive review by Cook and Wright [Ref. 14] in which alternatives to classical antibiotics are reviewed, antimicrobial blue light is not even mentioned in passing. It is in that context that we have sought to examine the effectiveness of blue light therapy of biofilm infections with the intent of ultimately translating this to clinical use when feasible, and specifically whether or not we could overcome some of the resistance to all types of therapy that biofilms represent by the use of an adjuvant therapy to increase the ROS formation that is the basis of phototherapy of infection.
While the mechanism of action of aBL is still not fully understood, the prevailing hypothesis is that endogenous chromophores in bacteria are able to absorb blue light and promote photochemical reactions. These photochemical reactions can be explained based on Jablonski Diagram in
In that context, bacterial wound biofilms are enclosed in an extracellular polymeric matrix, in which the relatively impermeable and often opaque matrix may partially explain their resistance to aBL eradication. Furthermore, in bacterial biofilms, bacteria tend to go into a metabolically dormant state, forming what are termed “persisters”, reducing their sensitivity to antibiotics, but also potentially to photo-reactivity. [Ref. 31]. For example, in our 48-hour in vitro biofilm model, vancomycin, a common antibiotic that is often used to treat serious MRSA skin infections, had only minimal effects on MRSA biofilms when combined with aBL and vancomycin was applied at an extraordinary concentration (1 mg/mL), which is 50 times above normally achievable serum levels (see
For many years, mean bactericidal concentration (MBC) has been defined as 99.9% killing of organisms, or 3 logs of killing [Ref. 24]. Both with antibiotics and with aBL, bacteria in biofilms have proven extraordinarily (100-10,000× more) resistant, and this level of killing has proven especially difficult with MRSA in biofilms. Ferrer-Espada et al. [Ref. 26] studied aBL in 48 hour monomicrobial and polymicrobial biofilms and found that a fluence of 216 J/cm2 of aBL (60 mW/cm2) was able to eradicate just 1.62 log10 CFU/mL of MRSA USA300 and 3.67 log10 CFU/mL of Pseudomonas aeruginosa.
Similarly, our findings demonstrate that aBL alone was not effective in eliminating more than 2 logs in MRSA biofilms in vitro, nor in the ex-vivo skin wounds. Therefore, we focused on exploring a strategy that enhances aBL activity through combining it with topical vitamin K3 (menadione) to increase the endogenous bacterial ROS production. Although there have been several studies representing the combination of aBL and adjuvants for MDR bacteria treatments [Ref. 17], this study represents the first that identifies Vitamin K3 (menadione) as a clinically viable strategy to potentiate aBL effectiveness in the killing of MDR bacteria, including MDR bacteria in biofilms, a major pathogenic mechanism in bacterial infections.
The synergistic effect on blue light ROS production was demonstrated when menadione was applied to the bacteria combined with aBL, showing a 2.5-fold increase of total ROS (see
It has been reported that menadione and some other quinones have analogous effects in generating reactive oxygen species through the catalytic reduction of oxygen with electrons that would have otherwise been involved in the electron transport chain in cellular respiration [Ref. 9]. The redox reaction generates reactive oxygen species through the reduction of oxygen and these ROS can induce oxidative damage to the bacteria. Schlievert et al. [Ref. 18] compared the ability of menadione to inhibit growth of S. aureus MN8 under anaerobic and aerobic conditions and found menadione was 4-fold more bactericidal for S. aureus when cultured aerobically. In the study, the author showed that the increased antimicrobial effect in the presence of O2 presumably reflects the important role that menadione plays as an inducer of redox cycling. In addition, another known effect of the blue light is a stimulation of electron transfer and proton motive force, that consequently increases both ROS and ATP production [Ref. 30] in bacteria. Without intending to be bound by theory, we hypothesize that the redox cycling activity of menadione is greatly augmented by the ROS generated by blue light. We further conclude that the ROS produced by menadione, combined with ROS generated by aBL, significantly increases bacterial intracellular oxidative stress, resulting in antimicrobial synergism.
We found an enhancement up to 104-fold in antimicrobial effects when the aBL was combined with menadione in MRSA and E. coli planktonic culture. Our findings further demonstrated that menadione synergistically potentiated the blue light antimicrobial activity even in the microbial biofilms (48 hours old). The effects of aBL were potentiated approximately 1000-fold when it was combined with menadione in MRSA and 100-fold in P. aeruginosa biofilms. The increase in effectiveness was also observed even with low power density of blue light in MRSA biofilms, an advantage in terms of tolerability in human tissue, heat dissipation and future clinical applicability to infected wounds. E. coli biofilms showed more resistance to treatment with the combination, but even these showed an enhancement of 1 log-fold killing in comparison with aBL alone.
We also studied the effects of menadione and aBL in bacterial biofilms in an ex vivo porcine wound model. The advantage of using a porcine ex vivo wound model is the similarity to human skin, enabling us to study a biofilm adhered to the skin, with high reproducibility and reasonable cost [Ref. 30]. An important finding was the significant potentiation of the antimicrobial effects by menadione when added to aBL in the ex vivo porcine wound model, which we are further investigating as a practical and inexpensive stimulant for screening candidate combination therapies for treating human skin wound biofilm infections with various forms of the light therapy. We found that the addition of low doses of topical menadione to aBL in the ex vivo model improved the aBL activity in MRSA by 104-fold and in P. aeruginosa 102-fold in comparison to aBL alone, consistent with our earlier in vitro biofilm treatment results, but now in a more challenging model.
Our findings demonstrate a significant potentiation of the antimicrobial effects of aBL with menadione in planktonic culture, biofilms and the ex vivo porcine wound model. Menadione alone did not kill bacteria in either the 48-hour biofilms in vitro or the ex vivo wound model, while showing some minimal antimicrobial activity against pathogens in planktonic culture as a single agent in the dark.
Menadione may produce other toxic effects to the bacterium besides direct oxidative damage. According to Andrade et al. [Ref. 8] due to its lipophilic character, menadione can interfere with bacterial membrane integrity, resulting in changes in membrane morphology and physiology and an increase of bacterial membrane permeability. In their study, the authors showed menadione led to an increase in aminoglycoside sensitivity by a membrane permeability mechanism in Staphylococcus aureus, but not against E. coli or Pseudomonas aeruginosa. In addition, Tintino et al. [Ref. 9] demonstrated inhibition of the norA gene by menadione. This is a critical antimicrobial resistance gene controlling the NorA efflux pump, and they demonstrated significant inhibition of both efflux pump gene expression and efflux pump function, restoring some degree of norfloxacin (fluoroquinolone) sensitivity, a potentially important clinically-applicable finding for treating surface wounds reachable by light and/or menadione.
In the absolute dark, menadione does not appear to generate significant ROS in bacteria. In bacteria exposed to blue light, menadione results in a substantial increase in ROS production by bacteria, by acting, our findings imply, as an exogenous photosensitizer to absorb photons and generate ROS as well acting as an ROS recycler. Combined with endogenous bacterial chromophores absorbing photons of antimicrobial blue light to generate endogenous ROS, which menadione regenerates, this results in a potentially toxic load of ROS to the bacteria, causing damage to bacterial membranes, efflux pumps, and bacterial DNA. The redundant antioxidant systems of mammalian cells are better protected, but as this therapy is advanced, this will have to be carefully examined.
Interestingly, Zheng and colleagues [Ref. 25] have demonstrated that menadione can act as a selective, potent inhibitor of the NLRP3 inflammasome, a multi-protein complex the activation of which, in humans, results in intense inflammatory reactions such as gout. The simultaneous improvement of antimicrobial activity with the potential for some local anti-inflammatory activity could prove quite beneficial in the topical treatment of wounds clinically.
In conclusion, we have demonstrated that the addition of menadione synergistically potentiated the effects of aBL, increasing bacterial oxidative stress to significantly increase microbicidal killing in a synergistic pattern in MRSA and P. aeruginosa, while having less effect on E. coli, less endowed with endogenous chromophores. In addition to enhancing ROS production by aBL, menadione has other effects, such as stimulating electron transport, inhibition of critical bacterial efflux pumps, and increased permeability of the bacterial membrane.
Our findings teach that the combination of aBL and menadione can be an effective alternative clinical strategy for the treatment of antimicrobial drug-resistant (AMR) bacteria-infected wounds capable of being reached by antimicrobial blue light. This is especially applicable to chronic wounds infected by AMR bacteria by potentially avoiding the use of antibiotics for the treatment of biofilm infections that often require prolonged courses of antimicrobial therapy. These pre-translational studies suggest that menadione can further amplify the utility of a “topical” therapy, antimicrobial blue light, with the potential to avoid further development of antimicrobial resistance, while providing synergistic “log-killing” of AMR bacteria in biofilm infections.
In conclusion, we have demonstrated that the addition of menadione synergistically potentiated the effects of aBL, increasing bacterial oxidative stress to significantly increase microbicidal killing in a synergistic pattern in MRSA and Pseudomonas aeruginosa, while having less effect on E. coli, less endowed with endogenous chromophores. In addition to enhancing ROS production by aBL, menadione has other effects, such as stimulating electron transport, inhibition of critical bacterial efflux pumps, and increased permeability of the bacterial membrane. We have shown that antimicrobial adjuvants (e.g., menadione) can be used in conjunction with antimicrobial blue light to reduce the bacterial bioburden in biofilms.
The citation of any document or reference is not to be construed as an admission that it is prior art with respect to the present invention.
We developed a prototype antimicrobial blue light bandage that is low power, potentially battery operated and thus portable, and capable of providing microbial suppression and microbial killing for prolonged periods of time using battery power in the field. Our device was made of inexpensive components that are easily scaled for mass manufacture. The device: (1) uses inexpensive LEDs at low power; (2) is low power, thus enabling battery-powered; (3) is specifically designed for low cost, mass manufacture; and (4) is light weight for use in the field by military, ERs, ambulances etc.
Existing U.S. military guidelines for the prevention of infections associated with combat related injuries include bandaging with a sterile dressing and transportation to a higher level of care. Administration of antibiotics at the point of care is advised according to the guidelines, especially if evacuation is delayed. It is anticipated that in the future, evacuation will become increasingly problematic. There is thus a need to stabilize and maintain a reduced bacterial load in wounds in the field for prolonged periods during which time continued antibiotic administration may not be optimal. We thus developed a prototype wound dressing capable of delivering prolonged antimicrobial activity, for one or more hours, days, weeks, months, or years. Specifically, we applied the use of antimicrobial blue light (aBL), which has shown promise as a nonantibiotic approach to combat microbial infections. ABL uses irradiation with visible light in a wavelength range of 400-470 nm and the production of reactive oxygen species (ROS), highly cytotoxic against bacteria. Phototherapy has been demonstrated to have potent antimicrobial properties against a broad range of microbes, including gram-positive and negative bacteria, mycobacteria, fungi and biofilms, demonstrated in in vitro, animal studies and at least one clinical trial. The advantages of local aBL treatment, compared to systemic antibiotics, include highly specific local targeting of bacterial burden, reduced risk of developing antimicrobial multi-drug resistant (MDR) infections, and avoidance of systemic side effects.
We first established desired features for a dressing in some use environments: lightweight and portable; low power for battery operation; easily mass manufactured from inexpensive components; biocompatible; light-transmitting; non-fouling; and highly bactericidal. The device may be used for initial treatment of a wound to prevent the development of a high bacterial wound burden and/or as treatment for an already contaminated wound. Inexpensive blue light (e.g., 405 nm) LEDs are located in a covering above the bandage, which sits in contact with the wound. We replicated this design for initial in vitro testing of bactericidal activity, varying power density, time of exposure, and thus radiant exposure. Bacterial viability was performed in planktonic and biofilm cultures of methicillin resistant Staphylococcus aureus (MRSA), methicillin-susceptible Staphylococcus aureus (MSSA), and Pseudomonas aeruginosa using aBL transmitted through the prototype bandage. Bacteria were quantified by counting colony-forming units/mL (CFU/mL). Different power densities (15, 30, 50 and 60 mW/cm2), time exposures and thus radiant exposures (radiant exposure=power density×time in seconds) of aBL were evaluated (0-720 J/cm2). Bactericidal activity was quantitated by reduction of CFU/mL of viable bacteria in both planktonic culture and in biofilms. Staphylococcus aureus suspensions containing 108 CFU/mL were exposed to continuous-wave 405 nm light for different power densities and radiant exposures. The monomicrobial biofilm was grown by inoculating 106 CFU/mL for MSSA, MRSA and Pseudomonas aeruginosa in 96-well microtiter plates. An issue using phototherapy is potential overheating on the skin caused by long periods of light exposure and power consumption for a portable device. To address these issues, we additionally studied the effects of varying the illumination duty cycle (50%; 10 seconds time on and 10 seconds off; or continuous-wave, 100%).
For MSSA planktonic culture, following 432 J/cm2, with continuous-wave 405 nm light through the bandage at power densities of 60, 30 and 15 mW/cm2, bactericidal activity was log reduced by 6.09, 4.67 and 2.84 CFU/mL, respectively. MSSA and MRSA were exposed to 405 nm light, duty cycle 50% at 50 mW/cm2, radiant exposure 360 J/cm2, resulting in log reduction of 5.05 for MSSA and 4.43 CFU/mL for MRSA. Temperature inside the target microtiter wells after continuous-wave 8-hour blue light treatment was 38.2° C., while the maximum temperature reached 27.8° C. after 24-hour with 50% duty cycle. We investigated 405-nm ABL inactivation of biofilms using the prototype device with 50% duty cycle. For 24-hour old monomicrobial biofilms formed in 96-well microtiter plates, 3.28-log CFU inactivation of MSSA and 1.87-log CFU inactivation of MRSA were observed after an ABL exposure of 720 J/cm2 through the bandage, at 50 mW/cm2, with 8-hour exposure time. However, when power density was decreased to 15 mW/cm2 with an increase of exposure time to 24 hours, (radiant exposure 648 J/cm2) the 24-hour old MRSA biofilm underwent a 2.72-log CFU inactivation while Pseudomonas aeruginosa underwent 5.77-log inactivation.
We developed a prototype antimicrobial blue light bandage that is low power, potentially battery operated and thus portable, and capable of providing microbial suppression and microbial killing for prolonged periods of time using battery power in the field. Our device is made of components that are easily scaled for inexpensive manufacture. We tested our prototype device by measuring in vitro killing of MSSA, MRSA, and Pseudomonas aeruginosa in both planktonic and biofilm cultures. We measured the effects of varying light radiation dosimetry parameters, including power density (mW/cm2) and radiant exposure (J/cm2), as well as duty cycle. The dissipation of heat is a major advantage of a duty cycle over continuous wave, particularly for a burn application. When the prototype aBL bandage was tested in vitro against planktonic MSSA, MSRA, and Pseudomonas aeruginosa approximately 3 logs or more of bactericidal activity are achieved. Similar bactericidal activity can be achieved with bacterial biofilms at low power densities using prolonged exposure times, dissipating heat with a duty cycle, resulting in high radiant exposures.
A prototype wound dressing capable of delivering prolonged antimicrobial activity by the dressing for as long as 7 days was developed. Specifically, we sought to apply the use of antimicrobial blue light as a non-antibiotic approach to reducing the bacterial burden in combat wounds. Antimicrobial blue light within the range of 400-470 nm has been demonstrated to produce reactive oxygen species that are highly cytotoxic against a broad range of microbes, including gram-positive and gram-negative bacteria, fungi, mycobacteria and biofilms containing these organisms. The advantages of local antimicrobial blue light treatment, compared to systemic antibiotics, include high specificity for microbes with correspondingly low cytotoxicity to mammalian cells, reduced risk of developing antimicrobial multidrug resistant (MDR) infections, and avoidance of systemic side effects seen with antibiotics. However, there was no available, portable antimicrobial blue light wound dressing for field use.
To address this military need, we have developed a prototype surface wound dressing, which we have termed the Photonic Antimicrobial Wound Surface Dressing (PAWS-Dressing), and evaluated and refined the prototype.
We first established criteria for an example embodiment or configuration of such a dressing: biocompatible; light-transmitting; highly bactericidal; non-fouling; lightweight and portable; low power for battery operation; made of inexpensive components for mass manufacture and disposability. We envisioned using the device for initial treatment of a wound to prevent the development of a high bacterial wound burden and/or as treatment for an already contaminated wound.
Based on this, we developed a prototype device using a 3-D printer and cast polydimethylsiloxane (PDMS) to be used as a dressing. PDMS is an FDA approved for use in disposable soft contact lenses, and as such is highly surface biocompatible, hydrophobic, oxygen permeable, breathable, highly translucent, flexible and moldable to fit the desired target area. PDMS is also inexpensive. Blue light (405 nm) LEDs were located above the PDMS bandage in a silicon LED-holder.
Immediately beneath the LEDs were inexpensive plastic convex lenses which uniformly distribute the light to the wound site. The PDMS bandage sits in contact with the wound, suspended off the wound by, in this example embodiment, about 2-8 millimeters (e.g., about 5 millimeters) on flexible PDMS “feet”, which enable gentle suction of exudate from the wound by an inexpensive Jackson-Pratt-like suction drain. We explored a number of patterns to optimize fluid flow and uniform light distribution. A wafer-thin, solid-state controller connected to a rechargeable portable battery sits on top of the LED holder. A clear Tegaderm-like adhesive tape holds the PDMS bandage on the wound, enabling gentle suction of exudate if present; the silicon LED holder can be secured to the surrounding uninjured skin with a standard adhesive bandage. Other issues when using phototherapy are potential overheating on the skin caused by long periods of light exposure and power consumption for a portable device. To address these issues, we additionally studied the effects of varying the illumination duty cycle (50% or continuous-wave; 10 seconds on and 10 seconds off).
We investigated other ways to enhance antimicrobial blue light to make it more widely useable against bacterial biofilms.
A bacterial suspension was grown in a shaking incubator overnight in BHI broth. Cells were collected by centrifugation at 4,000 rpm for 5 minutes and suspended in PBS at a density of 106 CFU/mL. Then, the biofilms were grown for 48 hours in 96-well microtiter plates with a renewal of the media every 24 hours. Following the 48-hour biofilm growth, fresh 200 μL of PBS with the tetracyclines minocycline, doxycycline, and demeclocycline in different concentrations were added to the wells and the biofilms were incubated in the dark at 25° C. for 1 hour: 0, 0.25, 0.5, and 1 μg/mL for minocycline; 0, 0.5, 1, and 2 μg/mL for doxycycline; and 0, 0.5, 1, and 2 for demeclocycline. After that, the control and the tetracycline-treated biofilms were maintained without irradiation (“menadione/dark”). Simultaneously, the aBL (405 nm) was delivered to the other biofilm groups testing different power densities (50, 30 mW/cm2) while keeping the radiant exposures of 50-250 J/cm2. After the light exposure, the biofilms were carefully washed two times with PBS and 200 μL of PBS was added to each well. Then, the bacterial biofilms in each well were harvested by scratching with a sterile pipette tip and three wells of each group were pooled together in a 1.5 mL microcentrifuge tube. The total volume obtained, 600 μL (3 wells), was sonicated using a Branson 2510 Water Bath Sonicator (Marshall Scientific, LLC) for 5 minutes and the CFU/mL was determined by 10-fold serial dilutions in PBS on BHI agar plates. CFU was counted after overnight incubation at 37° C.
We found an enhancement in antimicrobial effects when the aBL was combined with minocycline, doxycycline, or demeclocycline in MRSA biofilms (see
We investigated the possibility that minocycline and doxycycline might be acting as a photosensitizer. To do so, the production of ROS by minocycline and doxycycline combined with blue light was measured by the ROS probe DCF in MRSA. From
Thus, we have shown that antimicrobial agents (e.g., tetracyclines) can be used in conjunction with antimicrobial blue light to reduce the bacterial bioburden in biofilms. The use of tetracyclines in a wound dressing of the present invention has the potential to reduce the power consumption because when the light is off, there is still a residual antimicrobial effect to suppress bacteria.
Bacteria in biofilms are metabolically much less active (“dormant”, “persisters”) than planktonic bacteria, rendering them more resistant to antibiotics by 100-1,000-fold, and as we have seen, more resistant to oxidative killing by blue light. Near infrared (NIR-700 nanometers to 2000 nanometers) radiation of low power density can stimulate bacterial growth and energy metabolism. We demonstrated in this Example 5 that bacterial persisters/dormant bacteria in biofilms can be “turned on” by near infrared (NIR) of extremely low dose to become metabolically more active, generating intracellular ROS that can stimulate bacteria in biofilms (“dormant bacteria”, “bacterial persisters”).
We investigated the possibility that NIR radiation at 850 nanometers might be acting as a photosensitizer. To do so, the production of ROS by NIR at 850 nm at 10 mW/cm2 was measured by the ROS probes DCF and SOSG in MRSA biofilms. From
A bacterial suspension was grown in a shaking incubator overnight in BHI broth. Cells were collected by centrifugation at 4,000 rpm for 5 minutes and suspended in PBS at a density of 106 CFU/mL. Then, the biofilms were grown for 48 hours in 96-well microtiter plates with a renewal of the media every 24 hours. After that, the biofilms were: (1) maintained without irradiation (“control”); (2) irradiated with aBL (405 nm) at a radiant exposure 250 J/cm2; (3) irradiated with NIR at 850 nm to a radiant exposure of 1 J/cm2 and then irradiated with aBL (405 nm) to a radiant exposure 250 J/cm2; (4) irradiated with NIR at 850 nm to a radiant exposure of 10 J/cm2 and then irradiated with aBL (405 nm) to a radiant exposure 250 J/cm2; and (5) irradiated with NIR at 800 nm to a radiant exposure of 50 J/cm2 and then irradiated with aBL (405 nm) to a radiant exposure 250 J/cm2. After the light exposure, the biofilms were carefully washed two times with PBS and 200 μL of PBS was added to each well. Then, the bacterial biofilms in each well were harvested by scratching with a sterile pipette tip and three wells of each group were pooled together in a 1.5 mL microcentrifuge tube. The total volume obtained, 600 μL (3 wells), was sonicated using a Branson 2510 Water Bath Sonicator (Marshall Scientific, LLC) for 5 minutes and the CFU/mL was determined by 10-fold serial dilutions in PBS on BHI agar plates. CFU/mL was counted after overnight incubation at 37° C. We found an enhancement in antimicrobial effects in MRSA biofilm when aBL was combined with near infrared (NIR) radiation (see
In Example 5, we have shown that bacterial persisters/dormant bacteria in biofilms can be “turned on” by near infrared (NIR) of extremely low dose to become metabolically more active, rendering the bacteria more susceptible to both antimicrobial blue light and antimicrobial agents.
Thus, the present invention provides methods and devices for improving healing behavior of a biological wound, kits for improving healing behavior of a biological wound, and methods for killing or inhibiting growth of microbes in a biofilm such as bacteria in a biofilm on or adjacent a biological wound. We have developed a Photonic Antimicrobial Wound Surface Dressing (PAWS-Dressing) surface wound covering which delivers antimicrobial blue light. We have shown that antimicrobial adjuvants (e.g., menadione, NIR) and/or antimicrobial agents (e.g., tetracyclines) can be used in conjunction with antimicrobial blue light to reduce the bacterial bioburden in biofilms. Using methods and devices of the invention, one can develop a suite of wound dressings capable of addressing different clinical needs (e.g., “dry” ischemic ulcers, bleeding wounds, deep tissue infections, etc.) employing different wavelengths and configurations.
In light of the principles and example embodiments described and illustrated herein, it will be recognized that the example embodiments can be modified in arrangement and detail without departing from such principles. Also, the foregoing discussion has focused on particular embodiments, but other configurations are also contemplated. In particular, even though expressions such as “in one embodiment”, “in another embodiment”, “in embodiments”, or the like are used herein, these phrases are meant to generally reference embodiment possibilities, and are not intended to limit the invention to particular embodiment configurations. As used herein, these terms may reference the same or different embodiments that are combinable into other embodiments. As a rule, any embodiment referenced herein is freely combinable with any one or more of the other embodiments referenced herein, and any number of features of different embodiments are combinable with one another.
Although the invention has been described in considerable detail with reference to certain embodiments, one skilled in the art will appreciate that the present invention can be used in alternative embodiments to those described, which have been presented for purposes of illustration and not of limitation. Therefore, the scope of the appended claims should not be limited to the description of the embodiments contained herein.
This application is based on, claims benefit of, and claims priority to U.S. Application No. 63/270,249 filed on Oct. 21, 2021, which is hereby incorporated by reference herein in its entirety for all purposes.
This invention was made with government support under one or more of Department of Defense agreements #2019A 00456, fund #235656, and grant number HU00011920056. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/078479 | 10/21/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63270249 | Oct 2021 | US |
