TREATMENT OF INFECTED WOUNDS

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. Section 119(e) of copending U.S. Provisional Patent Application Serial No. 61/734,703, titled "Laser- based Bacterial Disruption for Treatment of Infected Wounds," filed December 7, 2012, the contents of which are incorporated herein by reference. TECHNICAL FIELD

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. BACKGROUND OF THE INVENTION

Wound infections and infected traumatic wounds impose a major burden on the healthcare system. Treatment of infected wounds, both surgical and traumatic, causes prolonged hospitalization, leads to a possibility of sepsis, and dramatically increases the cost of patient care [16-17]. Recent studies estimate that 5-7% of all surgical wounds become infected and 5-7% of all open traumatic wounds require open therapy for management of infections.

Additionally, 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.

Current wound therapy is based on reducing bacterial bioburden, removing necrotic debris, and applying negative pressure to a wound [39-43]. Topical therapies include the use of antibiotics, silver-containing hydrogels, and growth factors to promote wound healing. However, several 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].

A strong contributing factor to bacterial persistence in vivo is the ability of bacteria to generate biofilms. 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]. In addition to helping bacteria mechanically adhere to a wound surface, biofilms prevent the ingress of white blood cells and also provide a chemical barrier to both antibiotics and antibodies [7, 28-32]. Studies have shown that sessile bacteria in these biofilms can withstand host immune responses and are much less susceptible to antibiotics compared to their non-attached individual planktonic counterparts [93- 96]. For example, it has been shown that bacteria present in mature bio films are resistant to 50-5000 times the concentrations of antimicrobial agents that are necessary to kill planktonic cells of the same organism [27, 33]. Thus, current wound therapies are significantly less effective due to the existence of bacterial biofilms, which severely impact the normal wound healing mechanism, support bacterial resistance, and prevent the diffusion of antimicrobial agents into the bacteria.

Therefore, successful management of infected wounds requires the disruption of biofilms in order to increase bacterial vulnerability and reduce bioburden. If the aggregation of bacterial cells in biofilms can be broken down, host defenses and normal wound healing mechanisms may be able to resolve the infection and restore the efficacy of antibiotics [105]. There is widespread acceptance that a bioburden under 105 cfu/g of viable wound tissue is necessary for the normal healing process to continue [36-38]. With the treatment of biofilm-based infections costing more than $1 billion annually [99-101] and it being estimated that biofilms are associated with 65% of nosocomial infections [97-98], there is great interest in developing successful strategies for treating biofilms.

Several strategies for treating biofilms associated with wounds are currently being developed [104]. Wound debridement is a commonly used mechanical approach for biofilm disruption and reducing necrotic tissue and bioburden. For example, 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]. However, 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]. Thus, the lack of efficacy, need for repeated procedures, and subsequent patient discomfort has driven the development of alternative mechanisms of biofilm disruption and reduction of bioburden to treat chronically infected wounds. Other wound care treatments include chemical treatments and negative pressure wound therapies. However, chemical and negative pressure wound therapies have also been shown in studies to be relatively ineffective in treating bacterial wound infections. Chemical therapies include dissolving the matrix polymers of the biofilm using enzymes [106] and initiating chemical reactions that block biofilm matrix synthesis [107]. These chemical treatments have been shown to be effective in the short term, but are unable to completely eradicate biofilms, thereby allowing bacteria to ultimately proliferate again [45-48]. Furthermore, in another study, negative pressure wound therapy was found to actually increase bacterial colonization significantly in twenty- five patients [49].

Ultrasound therapy [111-112] and extracorporeal Shockwave therapy (ESWT) [113-114] have also been explored as wound care treatments, though each is associated with limitations. Therapeutic ultrasound (frequency range 0.75-3 MHz) are acoustic waves that propagate through mediums at frequencies greater than 20 kHz. Previously, ultrasound waves (high frequency, low wavelength) have been used in the low energy observation of tissues and organs. 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]. However, the application of ultrasound at low-intensities used in acoustic enhanced antibiotic killing does not change the shape of the biofilm nor does it alter the arrangement of the bacterial cells within the biofilm [8, 119]. Therefore, ultrasound therapy has been found to be ineffective on its own [50, 52-54] and incapable of delaminating biofilms [55]. Moreover, in some cases it has been found that low intensity ultrasound actually leads to an increase in bacterial cell growth [120]. Additionally, therapeutic ultrasound can create standing waves as a result of interference between reflected and incoming waves, giving rise to thermal generation that is detrimental to host tissues [1, 56-57]. Still further, the piezo-electric transducers that are used to generate the ultrasonic energy for breaking up bacterial bio films are cumbersome and expensive.

Extracorporeal Shockwave therapies (ESWT) have also been explored for wound healing. Similar to shock wave lithotripsy (SWL), which is used to break down kidney stones [2], 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. One study showed that ESWT reduced staphylococci bacterial growth (in vitro) after a set number of impulses at a specified energy flux density [6]. However, such 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.

Thus, there is a need in the art for wound therapies and mechanisms for disrupting biofilms and reducing bioburden in order to, for example, treat chronically infected wounds. In particular, there is a need for methods that can effectively disrupt and delaminate biofilms with fewer impulses than extracorporeal Shockwaves, while still remain safe with regards to mammalian cells and tissues [49, 51, 52, 59-65]. The present invention satisfies this and other needs. SUMMARY OF THE INVENTION

The instant disclosure provides methods and systems for decreasing bacterial bioburden and accelerating wound healing. As discussed below, 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. In typical methods of the invention, 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. In certain embodiments of the invention, the methodological parameters are controlled so that the peak stress of the Shockwave that is produced is greater than or equal to 50 MPa. In illustrative embodiments of the invention, 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. Typically in such methods, the laser absorbing composition comprises a flexible material coated with a metal. Optionally for example, the laser absorbing composition may comprise a polycarbonate, polyimide or polyester substrate coated with titanium and/or silver nanoparticles. In certain instances, 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. In one illustrative embodiment of the invention 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. In this illustrative method, 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. In this method, 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. In such methods, 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. In the working embodiments disclosed herein, the pulsed laser is a Nd:YAG laser with a wavelength of 1064 nm, and 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. Typically, 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. In this system, 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. In certain embodiments, 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 of the invention, the system comprises one or more elements designed to control, record, and/or characterize aspects of laser irradiation. For example, 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. Typically, 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.

Other objects, features and advantages of the invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the invention are given by way of illustration and not limitation. Many changes and modifications within the scope of the invention may be made without departing from the spirit thereof, and the invention includes all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS 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.

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.

FIG. 4 illustrates a (A) schematic design of a bench-top system using a Mylar substrate, according to one or more embodiments of the invention. (B) Characterization of stress profiles at various laser fluencies using a glass substrate. (C) Peak stress versus fluence using a glass substrate. FIG. 5 illustrates a portable cart system, according to one or more embodiments of the invention. (A) CAD design. (B) Photograph of assemble system. (C) Characterization of various flexible substrates used with the portable system.

FIG. 6 illustrates photomicrographs of S. epidermidis biofilm shocked using a glass substrate with various laser fluences: (A) control (B) 100 mJ (C) 200 mJ (D) 300 mJ (E) 400 mJ and (F) 500 mJ. Scale bars = 1 mm.

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 μιη. Abbreviations: 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.

FIG. 14 illustrates fluorescence images showing the effect of laser Shockwaves on mammalian cells (fibroblasts).

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention.

The present disclosure references a number of different publications as indicated throughout the specification by one or more reference numbers within brackets, e.g., [x]. A list of these different publications ordered according to these reference numbers can be found below in the section entitled "REFERENCES". Each of these publications is incorporated by reference herein.

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. In one LGST study, 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. Notably, Krespi et al. (2008) disrupted Pseudomonas biofilms on culture plates in vitro [75], while Nigri et al. (2001) showed no effect of LGST alone on microbial cell viability in vitro [76]. As discussed in detail below, 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. 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. In such embodiments, the Shockwave is controlled so as to break up the bacterial biofilm, while simultaneously stimulating antibiotic penetration of the biofilm. In addition, 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. Depending upon the severity of the infection, the process can be repeated multiple times. Similarly, 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).

Other embodiments of the invention include systems designed to disrupt bacterial bio films. Typically the system comprises a laser absorbing composition for contacting the bacterial biofilm, a laser for irradiating the laser absorbing composition. Optionally the system includes a portable cart housing the laser (e.g. a wheeled cart designed move from room to room in a clinical environment). In such systems, 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. In typical embodiments, 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. Typically, 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.

In embodiments of the invention, 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. In one embodiment, 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. In one exemplary implementation, the substrate comprises a thin layer (0.5 μιη) of titanium sandwiched between a 50- ΙΟΟμιη thick layer of waterglass and a 0.1 mm thick sheet of Mylar. In other embodiments of the invention, the surface of the substrate can be used as the laser- absorbing composition.

In typical embodiments of the invention, 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. Specifically, the laser absorbing composition exfoliates upon absorption of the laser energy and launches a mechanical stress wave into the substrate volume. When used in would healing, 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). In one embodiment, 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. In certain embodiments, 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). Typically, a peak stress generated by the Shockwave is between 50 MPa and 150 MPa.

In embodiments of the invention, the compressive stress waves generated are made incident on the biofilm-wound interface. In such embodiments, 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]. In this way, 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. On the contrary, the Shockwaves stimulate mammalian cells to release growth factors and produce collagen, which promotes healing. Thus, in contrast to ESWT, since 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]. However, due to the lack of direct interaction between the laser and skin, LGS does not result in similar damage. Also 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. In such methods, a laser absorbing composition can be applied to the wound (e.g. nanoparticle silver-coated plastic sheet). In certain embodiments, 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. Experiments have demonstrated that the laser-generated Shockwave is able to delaminate bacterial cells. As shown in Figure 14, most of the cells by a laser-generated Shockwave are dead, but the majority of cells around the shocked region are alive. The Shockwave also stimulates the cells to generate collagen and growth factors which promote healing. Additionally, the Shockwaves release any antibiotics in the antibiotic compound and promote the diffusion of the silver nanoparticles and/or antibiotics into the wound, helping eliminate subsurface bacteria. In specific examples, the pulsed laser is a Nd:YAG laser with a wavelength of 1064 nm, and 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.

In addition to biofilm disruption, 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]. Studies have found that LGS results in reduced bacterial viability when coupled with antibiotics [72-73]. The mechanism for this is thought to be related to the stress wave gradient which has a greater effect on cell viability than the peak stress of the Shockwave [74]. This provides evidence that a rapid stress rise-time (e.g. approximately 2-6 nanoseconds) should maximize cell wall permeability to allow maximum drug delivery.

Typically, 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). In certain embodiments of the invention, 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. For example, in one embodiment, a method for disrupting bacterial biofilm comprises generating a first mechanical shock wave to disrupt the bacterial biofilm. After cleaning the wound, 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 combination of biofilm disruption and therapeutic agents can dramatically reduce bacterial numbers, allowing the body to fight the infection.

In some embodiments of the invention, the stress wave is directly generated at the surface of the wound by removing the intermediate stress wave generating composition/substrate discussed above. In illustrative embodiments, 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. In 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. In 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. In 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. In 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. In step 6, if significant debris is generated, the gel is washed away or wiped off and the process can be repeated. In step 7, 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.

As shown in Figure 12, 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.

EXAMPLES

Using both a bench-top laser system to generate Shockwaves using a glass slide substrate coated with Titanium (Ti) and a portable laser system using flexible substrates, the examples disclosed herein show efficacy in delamination of biofilms from a variety of surfaces, safety of use on ex vivo porcine samples, and safety of use in mammalian cell lines. 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. 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.

Example 2: Portable Cart System

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).

In contrast to the bench-top system, the portable cart system utilizes a different substrate for laser impingement. In the bench-top system, a glass or polystyrene substrate, coated with Ti, was used to generate the Shockwaves. However, the rigid nature of these materials rendered their use on the 3 -dimensional contours of the preclinical or clinical subject fairly impractical. Using the portable cart laser system, 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). Using the portable cart laser system combined with the polycarbonate substrate coated with Ti, the efficacy in delamination of biofilms from a variety of surfaces including polystyrene, PDMS, and porcine samples overlaying ballistic gel phantoms of flesh was demonstrated.

Example 3 : Efficacy Experiment

The efficacy of the LGST has been investigated in both in vitro and ex vivo experimental settings using both the bench top system (see Example 1) and the portable system (see Example 2). In early experiments using the bench top system using a rigid glass substrate, it was demonstrated that LGST effectively delaminates bio film, and can reduce the number of living bacteria by up to 70% [77-79]. Experiments were performed on biofilms of Staphylococcus epidermidis grown in polystyrene dishes and irradiated with a range of laser fluences from 100-500 mJ focused on a 3 mm spot size (see Figure 13). Under these conditions, biofilms were delaminated at energy fluences greater than 300 mJ (Figure 6).

Delamination area increased substantially as energy fluence increased. Further investigation into bacterial viability after LGST revealed marked bacterial death (Figure 7). Live/Dead staining assays were performed on S. epidermidis using green fluorescent SYTO 9 stain for live bacteria, and red fluorescent propidium iodide stain for dead bacteria. As shown in Figure 7, much greater fluorescence of the red (dead) stain was found in the shocked region of a sample in comparison to the controls, indicating an increase in bacterial death with increased energy fluence.

In addition, the use of the bench top system was explored with a flexible

Mylar substrate. The use of a flexible substrate is of particular importance for translation into clinical trials as the use of a rigid substrate over in vivo wound beds is not practical. To this end, delamination of biofilms grown on PDMS tissue phantoms was demonstrated and further shocked using a Mylar substrate coated with Ti (Figure 8). Here, delamination was generated at even the lowest laser fluences, and again an increase in area of delamination was found with increasing energy.

Finally, ex vivo efficacy experiments were performed with the portable system. As mentioned above the portable laser system is being used with a polycarbonate substrate as this material was shown to yield the highest stress peaks amongst the flexible substrates of interest (Figure 5C). In these experiments, biofilm was grown on ex vivo porcine specimens over ballistic gel phantoms designed to emulate human flesh. Specimens were shocked over a similar range of laser fluences (100-500 mJ) over a 3 mm spot size. Specimens were evaluated using both paraffin histology, stained with a Gram positive stain for bacteria, and Scanning Electron Microscopy (SEM). The results reveal that successful delamination was possible at all energy fluences (Figure 9).

Therefore, the efficacy of the LGST system disclosed herein has been demonstrated. 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).

Example 4: Safety Experiment 1

In addition to efficacy, the safety associated with LGST was explored in vitro. Of particular concern are the thermal and mechanical effects of the Shockwave treatment on the epidermis and the underlying collagen structure of the dermis. Further, given the use of a laser, it is not surprising that concerns persist based on knowledge of laser damage to biological tissues when using other laser-based treatments. Some of these deleterious effects include ablation of the superficial layers of the skin, congealing of connective tissue, and tears or mechanical trauma in the various layers of the skin [80-84]. However, it is important to note that since the laser impinges on the metal coated substrate, the resulting mechanical Shockwave, and not the laser energy itself, comes into contact with the tissue. Therefore, laser-related tissue damage will not occur, since there is no direct contact between the laser and the tissue.

To test this concept, a safety study was conducted to quantify the effect of LGST at both the cellular and tissue levels of mammalian skin. An in vitro exploration was conducted into the viability of mammalian cell populations after LGST. Experiments were performed on mammalian fibroblasts divided into five experimental groups associated with five levels of laser fluence: 100, 200, 300, 400, and 500 mJ of energy. In addition, two control groups were included. The first was an alive control group where cells were not treated with any laser energy, and allowed to proliferate continuously. The second was a dead control group that was treated with methanol and killed. Cell metabolic activity was measured based on fluorescence values of all wells before treatment, 1 hour after treatment, and 24 hours after treatment. All samples were incubated between measurement points. 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.

Statistical analysis revealed that there was an initial decrease in fluorescence values due to LGST in mammalian cells, however, the cells quickly recovered and proliferated within 24 hours. Initial analysis showed no significant difference between groups prior to treatment (p = 0.067). One hour after samples were treated either with LGST or methanol, a drop in fluorescence was seen when compared to the alive controls. The dead controls, predictably, result in the lowest fluorescence values (p = 0.00001), while the various laser fluences show a more subtle, but still significant drop (Figure 10A, p < 0.0001). However, 24 hours after treatment, the LGST treated samples were significantly higher in cell viability than the dead controls (p < 0.0001), and several laser energy levels were not statistically significantly different than the alive control (p = 0.0176, 0.0356, and 0.096, respectively). Therefore, while a drop in mammalian cell viability occurred, the cell populations were strong enough to renew their numbers in 24 hours after treatment.

Example 5: Safety Experiment 2

Additional LGST safety studies were conducted on the tissue level to explore laser and mechanical damage to collagen structure in the epidermis and dermis. In this study, the effect of LGS on freshly harvested ex vivo porcine skin tissue samples was investigated. Materials and Methods

Specimen Preparation

The 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.

Substrate Preparation

Mylar sheets with dimensions of 8 x 3 cm by 0.1 mm thick were RF sputtered (Denton Discovery II 550) with 0.5 μιη 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 μιη. The waterglass layer acts as the constraining layer and is transparent to the Nd:YAG laser wavelength of 1.064 μιη.

Shockwave generating system and laser parameters 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 μιη Ti sandwiched between the back surface of the 0.1 mm thick Mylar sheet and the 50- 100 μιη 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].

Experimental Procedure

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.

Previous studies have shown that laser fluences between 100-500 mJ/pulse are capable of delaminating bio films off a variety of surfaces [124]. Thus, in this experiment, each pig skin sample was subjected to LGS of a particular laser fluence. Specifically, two tissue samples per energy level [118 (16.68), 149 (21.07), 228 (32.24), 264 (37.33), 350 (49.49), 400 (56.56), and 498 (70.42) mJ (mJ/mm ² )], and one control sample (N=15) were examined.

Sample preparation for analysis Immediately after shocking, the samples were fixed in formaldehyde and prepared for paraffin histology. Specimens were sectioned sagitally at 5μιη 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.

The 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. While assessing the overall appearance, 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.

Results

Qualitative observations of tissue sections under microscope

The tissue sections, including control, were viewed by an experienced pathologist in a comparative blind study. 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 stratum corneum, epidermis, dermis, and the epidermal-dermal junction were similar across all the irradiated specimens and control. There was no observable ablation of the top layers of the skin sample, congealing of the collagen fibers, or mechanical trauma in the various layers of the skin (Figure 10B). 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.

Tissue Section Scoring

The 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. If the slits/spaces were indeed caused by LGS then sample 498 'a' should have had the highest S score and 118 'a' and 'b' should have had the lowest S score. There is no clear pattern that can be observed in the S scores. This again suggests that the slits/spaces in the tissue sections are either inherently present in the tissue or are an artifact related to histology.

All the samples received a high O score of either 2 or 3. This indicates that the overall appearance of all samples was similar. In addition, no relationship between S and O scores and the energy level with which the samples were shocked can be observed. This indicates that treatment of the samples with LGS did not alter the overall appearance and structure of the ex vivo pigskin samples significantly.

Discussion

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.

Example 6: In vitro Model Experiment 1

Two strains of S. epidermidis NRS122 and NSR8 were plated on Tryptic Soy

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]. When 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. When 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.

Example 7: In vitro Model Experiment 2

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). In some embodiments, information on the exact layer at which the compressive wave rebounds as a tensile wave (i.e. where the tensile wave carries the greatest force and ability to delaminate) 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].

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CONCLUSION

This concludes the description of the preferred embodiment of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching. It is intended that the scope of the invention be limited not by this detailed description, but rather by the claims appended hereto.