WO2014089552A1 - Rupture bactérienne basée sur un laser pour le traitement de plaies infectées - Google Patents

Rupture bactérienne basée sur un laser pour le traitement de plaies infectées Download PDF

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Publication number
WO2014089552A1
WO2014089552A1 PCT/US2013/073842 US2013073842W WO2014089552A1 WO 2014089552 A1 WO2014089552 A1 WO 2014089552A1 US 2013073842 W US2013073842 W US 2013073842W WO 2014089552 A1 WO2014089552 A1 WO 2014089552A1
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laser
wound
absorbing composition
bacterial
biofilm
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PCT/US2013/073842
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English (en)
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David A. Haake
Vinay BHUPATHY
Artemio NAVARRO
Warren S. Grundfest
David O. BEENHOUWER
Vijay Gupta
Zachary D. TAYLOR
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The Regents Of The University Of California
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Publication of WO2014089552A1 publication Critical patent/WO2014089552A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/0613—Apparatus adapted for a specific treatment
    • A61N5/0624—Apparatus adapted for a specific treatment for eliminating microbes, germs, bacteria on or in the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/067—Radiation therapy using light using laser light

Definitions

  • the present invention relates generally to methods and systems useful in the treatment of infected wounds and in particular, the treatment of bacterial biofilm- infected wounds using laser-generated Shockwaves.
  • extremity wounds account for the majority of all combat-related injuries in the military. Penetrating and blast wounds generate extensive zones of injury that are contaminated with debris. These factors lead to a high incidence (up to 75%) of colonization and subsequent wound infections with bacteria and microorganisms from the environment. The occurrence of infection of these severely traumatized soldiers significantly increases their morbidity and delays their return to active duty. The emergence of highly resistant organisms (e.g. MRSA, Staphylococcus, and Acinetobacter) further complicates the management of infected wounds.
  • Normal wound healing is characterized by three overlapping phases: the inflammatory, proliferative, and remodeling phases [34]. If the body's vascular and cellular responses during the inflammatory phase are inadequate to overcome surface microorganisms and bacteria, the wound becomes predisposed to infection, delaying angiogenesis, tissue granulation, and re-epithelialization in subsequent stages, thereby making the wound chronic [35].
  • Chronic wounds are typically characterized by the formation of a coagulum, accumulation of necrotic debris, and leaked protein- containing fluids that serve as a rich medium for bacterial growth.
  • Topical therapies include the use of antibiotics, silver-containing hydrogels, and growth factors to promote wound healing.
  • species of bacteria including species from genera Staphylococcus and Acinetobacter, persist in infected wounds despite treatment with topical antibiotics, wound irrigation, and surgical debridement [19-23]. These bacterial strains often exhibit antibiotic resistance, which then leads to wound chronicity [18, 98, 102-103].
  • Bacterial biofilms are communal structures of microorganisms encased in an exopolymeric coat that attaches to both natural and abiotic surfaces [90-92].
  • Biofilms consist of a three-dimensional matrix layer rich with polymeric substances such as polysaccharides, nucleic acids, and proteins that provide a protective and nurturing environment for bacteria to proliferate and reside [24-27].
  • polymeric substances such as polysaccharides, nucleic acids, and proteins that provide a protective and nurturing environment for bacteria to proliferate and reside [24-27].
  • biofilms prevent the ingress of white blood cells and also provide a chemical barrier to both antibiotics and antibodies [7, 28-32].
  • Wound debridement is a commonly used mechanical approach for biofilm disruption and reducing necrotic tissue and bioburden.
  • burn wound bacterial endotoxin is known to play a major role in sepsis, and treatment normally includes aggressive and thorough debridement of the infected tissue [85-89].
  • debridement is often painful, time-consuming, and must be repeated frequently [44] since bacterial proliferation resumes immediately after the procedure is finished. Pain notwithstanding, wound debridement has not been sufficiently successful in dealing with bacterial resistance to antibiotic treatment [108- 110].
  • Therapeutic ultrasound frequency range 0.75-3 MHz
  • ultrasound waves high frequency, low wavelength
  • Some studies have subsequently found that low-intensity ultrasound may also have therapeutic effects that aid in wound healing and reduce bioburden when coupled with antibiotics [50-52].
  • In vivo studies on adult rats have shown that therapeutic ultrasound allowed for an acceleration of the initial stages of wound repair [4].
  • ESWT Extracorporeal Shockwave therapies
  • SWL shock wave lithotripsy
  • ESWT allows for focused, high amplitude pressure waves with non-thermal effects, thus allowing for an abrupt increase in energy over a defined area.
  • Three sources of generating acoustic Shockwaves include piezoelectric, electromagnetic and electrohydraulic sources [3].
  • Various types of skin lesions such as burns and ulcers have been tested by Shockwave therapy at low energy densities and high impulses. Results have shown that there was faster wound healing, which can be attributed to greater flow of blood in the affected area (i.e. angiogenesis) [5].
  • Ultrasonic Shockwaves have also been found to be detrimental to bacterial colonies.
  • ESWT reduced staphylococci bacterial growth (in vitro) after a set number of impulses at a specified energy flux density [6].
  • Shockwave therapies require a large number of impulses, up to 4000, and are susceptible to a cavitation-induced phenomenon that has been shown to further damage mammalian cells due to a tensile component of the mechanical stress wave [58, 121].
  • Cavitation bubbles are a non-thermal phenomenon that forms as the Shockwave passes liquid structures. The rapid expansion and collapse of these cavitation bubbles provide secondary local Shockwaves that aid in breaking down the kidney stones [2] and bio films but also damage mammalian cells.
  • inventions of the invention provide methods and systems for decreasing bacterial bioburden and accelerating wound healing.
  • embodiments of the invention include methods and systems for disrupting bacterial biofilms, fragmenting bacteria, and promoting the penetration of therapeutic agents into a wound.
  • the invention disclosed herein has a number of embodiments that relate to methodologies for disrupting bacterial biofilms.
  • a bacterial biofilm is contacted with a composition selected for an ability to thermally expand and generate a mechanical shock wave in response to laser irradiation.
  • This laser absorbing composition is then irradiated with a laser so that a mechanical Shockwave is generated in the bacterial biofilm, thereby disrupting the bacterial biofilm.
  • the methodological parameters are controlled so that the peak stress of the Shockwave that is produced is greater than or equal to 50 MPa.
  • the laser absorbing composition is irradiated by a pulsed laser where the pulse rise time is between 1/10 of a nanosecond and 10 nanoseconds.
  • the laser absorbing composition comprises a flexible material coated with a metal.
  • the laser absorbing composition may comprise a polycarbonate, polyimide or polyester substrate coated with titanium and/or silver nanoparticles.
  • the laser absorbing composition is coated with a layer of a transparent sodium silicate solution.
  • Methods of the invention can comprise generating a plurality of Shockwaves by irradiating the laser absorbing composition a plurality of times.
  • the methods can also include contacting the bacterial biofilm with at least one therapeutic agent, for example an antibiotic compound (e.g. a nanoencapsulated time-released antibiotic), and/or silver nanoparticles.
  • an antibiotic compound e.g. a nanoencapsulated time-released antibiotic
  • silver nanoparticles e.g. a first mechanical shock wave is generated to disrupt the bacterial biofilm; and a coupling medium comprising the therapeutic agent is then applied to the bacterial biofilm.
  • a second mechanical shock wave is then generated so as to promote penetration/diffusion of the therapeutic agent into the bacterial bio film, thereby further disrupting the biofilm and/or inhibiting bacterial growth.
  • a related aspect of the invention is a method for reducing bacterial bioburden and/or accelerating wound healing in a wound.
  • a laser absorbing composition is applied to the wound; a Shockwave is then generated at the wound by irradiating the laser absorbing composition with a pulsed laser.
  • the laser energy is controlled so that the biofilm in the wound is disrupted by the Shockwave while skin cells in the wound that are simultaneously exposed to this energy are not damaged by the Shockwave.
  • the pulsed laser is a Nd:YAG laser with a wavelength of 1064 nm
  • the laser absorbing composition is irradiated by the pulsed laser using a pulse rise time of between 1/10 of a nanosecond and 10 nanoseconds.
  • the conditions are controlled in order to generate a mechanical Shockwave having a peak stress greater than or equal to 50 MPa.
  • Embodiments of the invention also include systems for disrupting bacterial bio films.
  • Such systems typically comprise a laser absorbing composition for contacting the bacterial biofilm, a laser for irradiating the laser absorbing composition, and optionally, a portable cart housing the laser.
  • the composition disposed on the biofilm is selected for its ability to thermally expand and generate a mechanical shock wave in response to laser irradiation, one that disrupts the bacterial biofilm.
  • the laser is a pulsed laser selected to irradiate the laser absorbing composition with a pulse rise time of between 1/10 of a nanosecond and 10 nanoseconds.
  • the system comprises one or more elements designed to control, record, and/or characterize aspects of laser irradiation.
  • certain embodiments of the invention include a processor; a computer-readable program code having instructions, which when executed cause the processor to modulate one or more system parameters such as laser pulse rise time.
  • the system is designed to generate a mechanical Shockwave having a peak stress greater than or equal to 50 MPa.
  • the methods and systems disclosed herein are useful, for example in the treatment of resistant bacterial infections in patients. As the methods and systems disclosed herein can be used both for wound debridement and bacterial disruption, they are also particularly effective in the management of burn wounds and wounds with large amounts of necrotic debris. Consequently, the methods and systems disclosed herein can be used to accelerate wound healing and/or reduce to costs associated with the treatment of chronically infected wounds.
  • FIG. 1 illustrates a schematic of a laser-generated Shockwave treatment (LGST) system applied from the bottom-up, according to one or more embodiments of the invention.
  • LGST laser-generated Shockwave treatment
  • FIG. 2 illustrates a compressive wave, according to one or more embodiments of the invention.
  • FIG. 3 illustrates a schematic of a laser-generated Shockwave treatment (LGST) system applied from the top-down, according to one or more embodiments of the invention.
  • LGST laser-generated Shockwave treatment
  • FIG. 7 illustrates confocal photomicrographs of bacterial cell viability for (A) control and (B) laser-generated shocked biofilm specimens grown on a glass substrate. Dead bacteria fluoresce red, while live bacteria fluoresce green.
  • FIG. 8 illustrates photomicrographs of S. epidermidis biofilm shocked using a
  • Mylar substrate with various laser fluences (A) control (B) 100 mJ (C) 200 mJ (D) 300 mJ (E) 400 mJ and (F) 500 mJ.
  • FIG. 9 illustrates visualization of bacterial biofilm delamination from ex vivo procine samples.
  • A SEM of porcine sample with biofilm (rough areas) and shocked region indicated in red.
  • B Magnified view of the border between the delaminated shocked area (smooth region) and the unshocked area (rough region).
  • C Histological section of porcine sample after LGST. Sections were stained with a Gram positive bacterial stain. Bacteria can be seen stained black, and large black areas indicate biofilm on either side of the shocked region. The central, shocked region shows no biofilm, and very few individual bacterial cells.
  • FIG. 10 illustrates the safety study results described in Examples 4 and 5.
  • A Mammalian cell proliferation at various laser fluencies before, 1 hour after, and 24 hours after treatment.
  • B Histological sections of control and 498 mJ irradiated specimen showing no tissue damage and no observable differences between controls and treated samples. Specimens were sectioned sagitally, and stained with a Masson's trichrome stain. Scale bars are 100 ⁇ .
  • SC stratum corneum
  • E epidermis
  • D dermis.
  • FIG. 11 illustrates a schematic of a laser-generated Shockwave treatment (LGST) system, according to one or more embodiments of the invention.
  • FIG. 12 illustrates a schematic of a system for characterizing Shockwaves.
  • FIG. 13 illustrates (A) schematic of a system and (B) experimental procedure for studying the efficacy of LGST, as described in Example 3.
  • LGST laser-generated Shockwave treatment
  • FIG. 14 illustrates fluorescence images showing the effect of laser Shockwaves on mammalian cells (fibroblasts).
  • LGST in known in the art and has been shown to disrupt biofilms in vitro on various targets such as culture plates, sutures, and tympanostomy tubes [117]. Previous studies of LGST have produced mixed results.
  • a laser- based Shockwave generator was used to disrupt biofilms suspended in a water bath [24]. Pulsed Shockwaves with rapid rise times and nanosecond durations propagated a water medium and detached biofilm residue, while exposing the bacteria to external effects.
  • Nigri et al. (2001) showed no effect of LGST alone on microbial cell viability in vitro [76].
  • the invention disclosed herein includes new methods, methodological parameters, and systems for disrupting bacterial biofilms in order to, for example, decrease bacterial bioburden and accelerate wound healing.
  • These methods and systems utilize a laser-generated Shockwave treatment (LGST) in order to delaminate and/or disrupt biofilms from the environment in which the biofilm is adhered, for example, a wound surface in a patient.
  • LGST laser-generated Shockwave treatment
  • Embodiments of these methods can further be used to increase the penetration and efficacy of therapeutic regimens designed to address bacterial infections (e.g. by facilitating antibiotic contact with targeted microorganisms).
  • Embodiments of the invention include methods that apply a nanosecond pulsed laser energy to an infected wound, for example a wound that has a biofilm and which has been preliminary treated with nano-encapsulated time-released antibiotic- containing compounds and covered with a laser absorbing composition.
  • the Shockwave is controlled so as to break up the bacterial biofilm, while simultaneously stimulating antibiotic penetration of the biofilm.
  • the methodological parameters can be controlled so that the Shockwave stimulates the generation of collagen and other growth factors in the cells, phenomena which help facilitate the healing process.
  • the process can be repeated multiple times.
  • the process can also be tailored to facilitate wound debridement.
  • Embodiments of the invention include methods for disrupting a bacterial biofilm that include the steps of contacting the biofilm with a laser absorbing composition selected for its ability to thermally expand and generate a mechanical shock wave in response to laser irradiation. In these methods, the laser absorbing composition is then irradiated with a laser so that a mechanical Shockwave is generated in the bacterial biofilm, thereby disrupting the bacterial biofilm.
  • Embodiments of the invention include those designed so that a biofilm is delaminated from a wound surface, followed by subsequent treatment steps that promote wound healing (e.g. treatment steps that include contacting the wound with one or more therapeutic compositions).
  • the system comprises a laser absorbing composition for contacting the bacterial biofilm, a laser for irradiating the laser absorbing composition.
  • the system includes a portable cart housing the laser (e.g. a wheeled cart designed move from room to room in a clinical environment).
  • the composition is selected for an ability to thermally expand and generate a mechanical shock wave in response to laser irradiation and the laser is used to irradiate the laser absorbing composition so that a mechanical Shockwave is generated to disrupt the bacterial biofilm.
  • the laser is a pulsed laser selected to irradiate the laser absorbing composition with a pulse rise time of between 1/10 of a nanosecond and 10 nanoseconds. In certain embodiments, the laser is a pulsed laser selected to irradiate the laser absorbing composition with a pulse rise time of less than or equal to 6 nanoseconds.
  • the system is designed to generate a Shockwave having a peak stress greater than or equal to 50 MPa (e.g. between 50 MPa and 150 MPa). In such systems, the portable cart is designed to be small and compact and may be battery powered.
  • the laser absorbing composition can comprise a substrate (preferably a flexible composition such as polycarbonate) coated with a metal.
  • the laser absorbing composition can, for example, comprise a glass, polycarbonate, polyimide or polyester substrate.
  • the substrate composition can be coated with a metal such as a layer of titanium and/or silver nanoparticles.
  • a short light pulse is focused onto a laser absorbing film that in turn is coated on the front surface of a substrate disc.
  • the laser absorbing composition may also be further coated and constrained with a layer of a transparent sodium silicate solution, such as a transparent waterglass layer.
  • the substrate comprises a thin layer (0.5 ⁇ ) of titanium sandwiched between a 50- ⁇ thick layer of waterglass and a 0.1 mm thick sheet of Mylar.
  • the surface of the substrate can be used as the laser- absorbing composition.
  • an electromagnetic radiation source such as a laser is used to impinge upon a laser absorbing composition, thereby generating mechanical Shockwaves due to the rapid thermal expansion of the medium.
  • the laser absorbing composition exfoliates upon absorption of the laser energy and launches a mechanical stress wave into the substrate volume.
  • the stress wave exits on the opposite side of the substrate it is coupled into the wound site where it interacts directly with the bacterial biofilm (e.g. one present on a wound surface).
  • ND:YAG laser energy is used to generate compressive stress waves through the rapid thermal expansion of a metallic thin film [123, 134].
  • the laser fluence, pulse width, and the substrate material properties contribute to the temporal characteristics of the stress wave.
  • the laser absorbing composition is irradiated by a pulsed laser such that a pulse rise time of between 1/10 of a nanosecond and 10 nanoseconds (e.g. less than or equal to 6 nanoseconds).
  • a peak stress generated by the Shockwave is between 50 MPa and 150 MPa.
  • the compressive stress waves generated are made incident on the biofilm-wound interface.
  • the wave upon encountering the interface, the wave reflects as a tensile wave from the biofilm's free surface, resulting in its spallation at sufficiently high amplitudes [124].
  • the laser energy is not directly incident on the tissue.
  • the generated stress wave in such embodiments directly breaks up the bacterial biofilm and disrupts the bacteria. Failure is both cohesive as well as adhesive at the interface. These local failures are caused by the shear and tensile stresses that are generated inside the biofilm by the propagating stress wave. However, the Shockwaves do not damage mammalian cell walls as they are more pliable and elastic.
  • the Shockwaves stimulate mammalian cells to release growth factors and produce collagen, which promotes healing.
  • the methods disclosed herein primarily use compressive stress waves to delaminate bio films [122], the damage caused by cavitation bubbles is eliminated. This allows faster rise times, shorter pulse durations, and no cavitation effects due to the lack of a tensile component interacting with the tissue.
  • the laser-generated Shockwave (LGS) treatment is unique in comparison to other techniques that involve treatment with laser energy. The difference arises from the fact that in other treatments, the laser is made incident on human skin directly. Some of the common deleterious effects that result from this direct interaction include ablation of the top layers of the skin [125, 126], congealing of connective tissue [127], as well as tears and trauma in the various layers of the skin [128].
  • LGS does not result in similar damage.
  • LGS has a very high strain rate loading (approximately 10 7 s "1 ) because of the short (sub-nanosecond) rise time. As a result, LGS suppresses the behavior of the tissue as an inelastic material. Thus, deformations in the structure of the tissue do not occur (see Examples 4 and 5).
  • Embodiments of the invention include methods for reducing bacterial bioburden and/or accelerating wound healing.
  • a laser absorbing composition can be applied to the wound (e.g. nanoparticle silver-coated plastic sheet).
  • a layer of a nanoencapsulated time-related antibiotic compound is also applied to the wound.
  • Laser pulses are then targeted to impact on the laser absorbing composition covering the wound in order to generate a Shockwave.
  • the biofilm in the wound is then disrupted by the Shockwave while host cells in the wound are not damaged by the Shockwave.
  • the laser-generated Shockwave is able to delaminate bacterial cells.
  • the pulsed laser is a Nd:YAG laser with a wavelength of 1064 nm
  • the laser absorbing composition is irradiated by the pulsed laser at a pulse rise time of between 1/10 of a nanosecond and 10 nanoseconds (e.g. less than or equal to 10, 9, 8, 7, 6 or 5 nanoseconds) and/or to generate a Shockwave having a peak stress between 50 MPa and 150 MPa.
  • LGST has also been shown to enhance the permeability of bacterial biofilms [66-67, 115-116], thereby facilitating the delivery of agents including macromolecules and genes through cell membranes and skin [68- 71].
  • the methods disclosed herein comprise of generating a plurality of Shockwaves by irradiating the laser absorbing composition a plurality of times. These methods can also comprise the step of contacting a bacterial biofilm with an antibiotic compound, such as a nanoencapsulated time-released antibiotic, and/or silver nanoparticles and/or an antiseptic compound such as a solution comprising a povidine iodine (e.g. Betadyne).
  • the method may be carried out as a two-step procedure where the biofilm is disrupted using a first stress wave; and then silver nanoparticles and antibiotics are propelled into a cleaned wound using a second stress wave.
  • a method for disrupting bacterial biofilm comprises generating a first mechanical shock wave to disrupt the bacterial biofilm.
  • a coupling medium such as a gel comprising an antibiotic compound (and or comprising a compound that promotes adhesion between a biofilm and a laser absorbing composition) is applied to the bacterial biofilm.
  • a second mechanical shock wave is then generated wherein the second mechanical shock wave promotes diffusion of the antibiotic compound into the bacterial biofilm.
  • the second stress wave is generated inside the substrate but is coupled into the wound site through the use of the coupling medium.
  • the stress wave is directly generated at the surface of the wound by removing the intermediate stress wave generating composition/substrate discussed above.
  • the wound surface is covered with a layer of nano-encapsulated antibiotics and then covered by another layer of silver nanoparticles on the top.
  • the silver nanoparticles in the top layer act as a laser absorbing composition to launch the stress wave directly into the wound.
  • the Shockwave propels both the silver nanoparticles and the antibiotics into the wound while also breaking up the bacterial biofilm and disrupting the bacteria.
  • An illustrative embodiment of the invention can be implemented as follows. In step 1, the wound is cleaned of surface debris.
  • step 2 a photograph of the wound is taken and this information is used to produce a plastic sheet with nano-encapsulated silver particles to cover the area of the wound. This sheet also outlines the area to be treated with laser pulses.
  • step 3 a handheld nanosecond-pulsed mid-infrared laser with a spot size of approximately 1 mm is rapidly translated within the borders outlined on the plastic sheet. Pulses are separated by approximately 1 mm and the repetition rate is approximately 500 pulses per second. The number of repeated scans depends upon the severity of the infection and the type of tissue to be treated.
  • step 4 a thin layer of gel containing the nanoparticle-encapsulated antibiotic is swabbed into the wound.
  • This layer provides acoustic coupling for the transmission of the Shockwaves and ensures that air does not interfere with Shockwave transmission.
  • step 5 after the gel is applied, the plastic sheet is placed on the wound, all air is expelled, and the laser pulses are allowed to impinge on the plastic sheet. If necessary, local anesthetic can be combined in the gel layer to numb the area.
  • step 6 if significant debris is generated, the gel is washed away or wiped off and the process can be repeated.
  • the wound is then dressed.
  • Figure 11 shows an illustrative embodiment of the invention.
  • a 2-axis galvo- mirror provides two-dimensional scanning of the wound area covered with biofilm.
  • a 1064 nm pulsed laser passes through and is focused by a focusing lens.
  • the laser irradiates a laser absorbing composition, in this instance a flexible aluminum/polyimide substrate.
  • a 1064 nm spot pattern of the wound area is traced out by the galvo-mirror.
  • the laser absorbing composition is layered on top of coupling medium comprising silver nanoparticles and nanoencapsulated antibiotics.
  • the coupling medium is layered over the bacterial biofilm.
  • the irradiated laser absorbing composition generates a Shockwave that propagates through the coupling medium to disrupt the biofilm.
  • the Shockwave can be characterized with a 632.8 nm HeNe probe.
  • the top side of a substrate material is metalized with an absorbing film and the bottom side of the substrate material is metalized with a reflecting film.
  • This coated substrate is used as a mirror in the moving arm of an interferometer. Interference fringes are recorded with a fast digitizer and the data is fitted to extract a stress profile.
  • Embodiments of the invention disclosed herein can increase the efficacy for treating infected wounds, reduces the need for systemic antibiotics, reduce the need for wound dressing changes, and shorten healing times. These improvements provide significant cost savings. Furthermore, embodiments of the system can be portable, handheld, compact, and easy to use.
  • Example 1 Bench-Top System
  • a large bench-top Nd:YAG laser was used to generate a 2-6 nanosecond pulse focused over a 3 mm diameter area on a 0.5um thick Titanium (Ti) film sandwiched between the back surface of a glass slide and a 50-100 um layer of Si0 2 (see, e.g. U.S. Patent 5,438,402). Titanium was used due to its high absorption coefficient with respect to the wavelength of the Nd:YAG laser [77].
  • the melting induced expansion of Ti under confinement generates a compressive stress wave with a sun-nanosecond rise time that then travels towards the underlying biofilm and tissue sample.
  • the compression stress pulse Upon interacting with the Ti coating, the compression stress pulse reflects into a compressive and tensile component.
  • the compressive component travels through the biofilm and down into the underlying tissue, while the tensile component reflects from the biofilm surface leading to spallation of the biofilm from the tissue sample.
  • the stress profiles has been successfully characterized using both glass substrates and polystyrene substrates to determine how much pressure is being delivered to the samples at various laser f uences ( Figure 4A-C). Using this system, successful delamination of biofilms from polystyrene surfaces, polydimethylsiloxane (PDMS) tissue phantoms, and ex vivo porcine tissues has been demonstrated.
  • PDMS polydimethylsiloxane
  • the portable cart system utilizes a Brilliant B (Quantel, France) closed
  • Nd:YAG laser (1064 nm) mounted, with its power source, onto a cart. It includes a translation stage to properly align the laser to a target of variable height, and is designed in such a way to allow LGST on live animal subjects ( Figure 5 A, B).
  • the portable cart system utilizes a different substrate for laser impingement.
  • a glass or polystyrene substrate, coated with Ti was used to generate the Shockwaves.
  • the rigid nature of these materials rendered their use on the 3 -dimensional contours of the preclinical or clinical subject fairly impractical.
  • stress profile experiments were performed using a variety of flexible substrates including polycarbonate, acetate, polyester, polyvinyl chloride (PVC), and polyether ether ketone (PEEK). This comparative study revealed that polycarbonate films were the best candidates for additional exploration (Figure 5C).
  • the system is capable of not only delaminating biofilms from a variety of substrates ( Figures 6, 8, 9), but is also quite effective at killing individual bacterial cells, and therefore reducing bioburden ( Figure 7).
  • LGST The effect of LGST on cell metabolic activity was tested using a repeated measures ANOVA analysis. Data collected at each time point was treated as a repeated measure. Each pair of treatments was then compared post hoc using a Tukey HSD multiple comparisons based on least-squares means at a Bonferonni-corrected significance level of 0.007.
  • porcine model was chosen as it has been used as a model for human skin in the past due to its similarities to human skin both physiologically and anatomically [128, 129].
  • Porcine skin specimens were harvested from the abdominal region of a pig immediately post-mortem. The specimen was cut into square shaped pieces of 5 mm length using a scalpel and blade. It was maintained at room temperature (25°C) throughout the experiment and not frozen so as not to alter the structure or mechanical properties of the collagen fibers. LGS treatment was carried out no more than 30 minutes after specimens were harvested.
  • Mylar sheets with dimensions of 8 x 3 cm by 0.1 mm thick were RF sputtered (Denton Discovery II 550) with 0.5 ⁇ of Titanium (Ti).
  • a uniform layer of waterglass (Si02) was then spin-coated on top of the Ti to achieve a uniform layer of 50-100 ⁇ .
  • the waterglass layer acts as the constraining layer and is transparent to the Nd:YAG laser wavelength of 1.064 ⁇ .
  • a Q-switched, ND:YAG laser was used to generate LGS.
  • a 3-6 nanosecond (ns) long Nd:YAG laser pulse is impinged over a 3 mm diameter area on the 0.5 ⁇ Ti sandwiched between the back surface of the 0.1 mm thick Mylar sheet and the 50- 100 ⁇ thick layer of waterglass.
  • Laser-generated pulses impinging upon the thin metallic surface generate stress waves within the material.
  • the laser energy ablates the thin metallic film, thereby causing a rapid thermal expansion of the irradiated titanium film, resulting in a transient compressive wave propagating through the substrate that is coupled through a liquid layer to the surface of the ex vivo pigskin sample.
  • the laser fluence, pulse width and the substrate material properties contribute to the temporal characteristics of the stress wave.
  • the peak stress, rise time of the wave, and the stress profile generated are dependent on the above mentioned parameters [130-135].
  • the pig skin sample was immersed in a petri dish containing DI water, such that there was a 1 mm thick layer of DI water over the pigskin.
  • the DI water was used as a coupling agent between the pigskin and the Mylar sheet.
  • the pigskin was held in place by placing it in between two acrylic blocks glued to the base of the Petri dish. The Shockwaves were made to be perpendicularly incident on the pig skin sample.
  • Sample preparation for analysis Immediately after shocking, the samples were fixed in formaldehyde and prepared for paraffin histology. Specimens were sectioned sagitally at 5 ⁇ along the midline, coinciding with the center of 3 mm shocked region. This region should correspond to the maximum mechanical impact due to the laser Shockwaves. The sections were then stained using H&E and Masson's Trichrome stain ( Figure 10B). The tissue sections were then analyzed using light microscopy.
  • tissue sections were scored on the basis of their overall appearance (O) and the presence of linear/slit-like spaces roughly parallel to the surface of the skin (S) (these spaces are probably an effect of LGS on skin) when compared with other samples, on a scale from 0 to 3.
  • a score of 0 indicated healthy tissue and 3 indicated maximal damage to tissue.
  • the tissue sections were examined to see whether the stratum-corneum, epidermis, dermis and the epidermal-dermal junctions were intact. Indications for ablation of the top layers of the skin, congealing of the collagen fibers and mechanical trauma to the various layers of the skin were looked into. It was also evaluated whether changes in the collagen structure and orientation took place.
  • the resulting scores indicate no observable differences between the control specimens and the irradiated specimens [84]. There was no relationship between the score received.
  • the collagen structure and orientation remained intact, and no differences could be observed when compared to the control sample. There were some regions where the collagen fibers seem to have larger spaces or air pockets in between them, but such regions were also found in the control sample indicating that it is most probably an artifact related to preparation of specimens or sectioning.
  • control samples were given the highest slit/space and overall score. This suggests that the slits/spaces seen in the collagen structure is most probably a sectioning or preparation artifact.
  • the high O score indicates that the control sample looks very similar to the other samples when compared on the basis of overall appearance.
  • 9 of the 15 samples e.g. Sample 149 'a', 228 'a' & 'b', 264 'b', 350 'a' & 'b', 400 'a' & 'b' and 498 'b' received a S score of 2. Furthermore, the samples 498 'a' and 118 'a' and 'b' received the lowest S score of 1.
  • LGS treatment does not have an adverse effect on the structure of pigskin. Histology demonstrates that at the energies tested various structural components of the skin; the epidermis, dermal-epidermal junction and the dermis, remain intact after subjecting the pigskin specimens to LGS. Additional studies showed that there was no change in the structure and orientation of the collagen fibers in the pigskin samples. This example provides evidence that LGS can delaminate bacterial bio films from skin without damaging the underlying skin cells.
  • Agar The bacteria were grown in colonies at 37°C for 24 hours. Biofilm was also grown directly on the polystyrene bottom of a petri dish by incubating 5 mL of Tryptic Soy Broth with an inoculation of overnight bacterial culture. The bottoms of the petri dishes used to grow the agar were coated by sputtered aluminum onto the polystyrene surface in a vacuum and then spin coated with waterglass.
  • a YAG laser was used to irradiate the bacteria as pictured in Figure 1.
  • the 7 nanosecond YAG laser pulse with a rise time of 2 nanoseconds had an energy of approximately 80 mJ. It was unaffected by the waterglass and passed straight through to the aluminum. When aluminum is irradiated, it goes through a 6.5% thermal expansion, but is constrained by the waterglass and the Shockwave generated is transmitted through the agar [11].
  • the Shockwave passed through the agar, it moved as a compressive wave, which did not delaminate the material but merely "pushed" the materials together as pictured in Figure 2.
  • the compressive wave reached a free surface it rebounded as a tensile wave. The tensile wave broke the adhesion between the bacteria and the agar/polystyrene thereby delaminating the biofilm.
  • Biofilm growth was performed in the same way as in the previous Example 6. All of the growth media to which the bacteria were added was supplemented with 4% EtOH, 2-4% NaCl, or 0.25%> Glucose. These stresses have been demonstrated to produce biofilms in S. epidermidis by Christensen et al, [12, 13].
  • a top-down approach required setup as pictured in Figure 3. In this setup, the laser pulse travels through the waterglass and creates a similar compressive wave. The difference is that this wave travels down through the layers (Tryptic Soy Broth(TSB), biofilm, agar, polystyrene dish) as opposed through up as in the previous experiment (see Example 6).
  • information on the exact layer at which the compressive wave rebounds as a tensile wave is used to modulate aspects of the invention.
  • the results were viewed under fluorescent microscopy, which revealed that pieces of the biofilm and bacteria were expelled into the solution [14, 15].
  • Extracorporeal shock wave treatment modulates skin fibroblast recruitment and leukocyte infiltration for enhancing extended skin-flap survival. Wound Repair Regen. 17: 80-87.

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Abstract

L'invention concerne des procédés et des systèmes pour le traitement de plaies infectées. Les procédés et les systèmes comprennent l'utilisation d'un laser pulsé nanoseconde. Appliqué à la plaie, qui a été prétraitée avec un composé antibiotique retard nanoencapsulé et recouverte d'une composition absorbant le laser revêtue d'argent, le laser irradie la composition absorbant le laser recouvrant la plaie et génère une onde de choc qui rompt le biofilm bactérien et libère l'antibiotique. En même temps, les ondes de choc favorisent la diffusion des nanoparticules d'argent et des antibiotiques dans la plaie, contribuant à éliminer les bactéries en dessous de la surface. Les cellules ne sont pas endommagées par les ondes de choc et sont stimulées pour produire du collagène et des facteurs de croissance qui activent la cicatrisation. En fonction de la gravité de l'infection, le procédé peut être répété plusieurs fois.
PCT/US2013/073842 2012-12-07 2013-12-09 Rupture bactérienne basée sur un laser pour le traitement de plaies infectées WO2014089552A1 (fr)

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US11617895B2 (en) 2015-07-28 2023-04-04 Know Bio, Llc Systems and methods for phototherapeutic modulation of nitric oxide
CN110831662A (zh) * 2017-05-08 2020-02-21 沃库尔医疗技术有限公司 用于伤口的处理的激光装置
WO2018206066A1 (fr) * 2017-05-08 2018-11-15 Vulcur Medtech Aps Dispositif laser de traitement de plaies
CN111032111A (zh) * 2017-08-18 2020-04-17 心脏器械股份有限公司 血泵中的治疗性紫外线血液处理
RU2737417C1 (ru) * 2019-11-27 2020-11-30 Федеральное государственное бюджетное учреждение науки Физический институт им. П.Н. Лебедева Российской академии наук (ФИАН) Способ борьбы с бактериальными биоплёнками
EP3881790A1 (fr) * 2020-03-15 2021-09-22 Martin Ivanov Denev Utilisation d'un choc mécanique photohydraulique pour la destruction sélective de membrane de cellules cancéreuses
US11147984B2 (en) 2020-03-19 2021-10-19 Know Bio, Llc Illumination devices for inducing biological effects
US11684798B2 (en) 2020-03-19 2023-06-27 Know Bio, Llc Illumination devices for inducing biological effects
US11752359B2 (en) 2020-03-19 2023-09-12 Know Bio, Llc Illumination devices for inducing biological effects
US11986666B2 (en) 2020-03-19 2024-05-21 Know Bio, Llc Illumination devices for inducing biological effects
US11654294B2 (en) 2021-03-15 2023-05-23 Know Bio, Llc Intranasal illumination devices
CN113425891A (zh) * 2021-07-08 2021-09-24 河北大学 负载光合细菌的水凝胶及其制备方法和应用
WO2024042447A1 (fr) * 2022-08-22 2024-02-29 Laserleap Technologies, S.A. Dispositifs et procédés d'amorçage de tumeurs solides avec des impulsions de pression pour renforcer des thérapies anticancéreuses

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