WO2008065652A2 - Génération de ros par les nanoparticules, microbulles et leur utilisation - Google Patents

Génération de ros par les nanoparticules, microbulles et leur utilisation Download PDF

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WO2008065652A2
WO2008065652A2 PCT/IL2007/001462 IL2007001462W WO2008065652A2 WO 2008065652 A2 WO2008065652 A2 WO 2008065652A2 IL 2007001462 W IL2007001462 W IL 2007001462W WO 2008065652 A2 WO2008065652 A2 WO 2008065652A2
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nanoparticles
ros
ultrasound
nanoparticle
microbubble
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PCT/IL2007/001462
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WO2008065652A3 (fr
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Hanoch Kislev
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Hanoch Kislev
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B18/00—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body
    • A61B18/18—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves
    • A61B18/1815—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves using microwaves
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/02—Radiation therapy using microwaves
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N7/00—Ultrasound therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N2/00—Magnetotherapy
    • A61N2/002—Magnetotherapy in combination with another treatment
    • 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
    • A61N2005/0658—Radiation therapy using light characterised by the wavelength of light used
    • A61N2005/0659—Radiation therapy using light characterised by the wavelength of light used infrared
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N7/00—Ultrasound therapy
    • A61N2007/0004—Applications of ultrasound therapy
    • A61N2007/0017—Wound healing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N7/00—Ultrasound therapy
    • A61N2007/0004—Applications of ultrasound therapy
    • A61N2007/0034—Skin treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N7/00—Ultrasound therapy
    • A61N2007/0039—Ultrasound therapy using microbubbles
    • 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
    • A61N5/062—Photodynamic therapy, i.e. excitation of an agent

Definitions

  • the present invention is of compositions for generating ROS (reactive oxygen species) and uses thereof, and particular such compositions and uses in which the ROS are generated through the application of nanoparticles and microbubbles.
  • ROS reactive oxygen species
  • Oxygen is vital to most human and animal life. It can, however, give rise to a variety of reactive oxygen species ("ROS") as part of normal metabolism. Reactive species are produced by the body under normal conditions, and indeed are part of normal metabolism. The body is equipped with a variety of mechanisms which render ROS inactive.
  • ROS reactive oxygen species
  • ROS are the byproducts of mitochondrial electron transport, various oxygen- utilizing enzyme systems, peroxisomes, and other processes associated with normal aerobic metabolism as well as lipid peroxidation. These damaging byproducts further react with each other or other chemicals to generate more toxic products.
  • hydrogen peroxide can be transformed to the highly reactive hydroxyl radical (OH) through various reactions.
  • Human beings have a defense system against toxic byproducts of metabolism including enzymes such as superoxide dismutase, catalases, peroxidases and antioxidants such as vitamins (e.g., vitamin A, beta-carotene, vitamin C and vitamin E).
  • Hydrogen peroxide can be transformed by catalases and peroxidases to oxygen and water. Thus, under normal conditions, the rate of ROS production does not exceed the capacity of the tissue to catabolize them.
  • ROS reactive oxygen species
  • ROS induce programmed cell death or necrosis, induce or suppress the expression of many genes, and activate cell signaling cascades, such as those involving mitogen-activated protein kinases.
  • Several enzymes have been recognized as being potentially able to produce ROS; however the most important of these is NADPH oxidase.
  • NADPH oxidase has been found in cells that 30 have no role in host defense. For example, NADPH oxidase components have been reported in fibroblasts, mesangial cells, endothelial cells, osteoclasts and chondrocytes.
  • ROS generation e.g., 10.sup.13 to 10.sup.15 ROS per cm.sup3
  • the cell-related activities of such light absorption are believed to include cell replication, cell metabolism, protein synthesis, ATP production, mitochondria replication, phagocytosis, and photodissociation of oxygenated hemoglobin (Kara, The Science of Low-Power Laser Therapy, Gordon and Breach, 1998, “Photobiology of Low Power Laser Effects", Health Physics, vol. 56, May 1989).
  • ROS generation induces tissue related effects, including: capillary formation, parasympathetic nervous system stimulation, increased endorphin release, increased production and release of adrenal steroids, immune system stimulation, enhanced fibroblastic production and collagen synthesis, and accelerated healing of wounds.
  • ROS can also alter the redox state of a cell which in turn alters cellular enzymes activity.
  • Extracellular ROS can affect Ca+2 channels and in turn modify the Ca+2 flow into the cell.
  • Ca+2 intake is responsible for cell growth, proliferation, differentiation, enhanced metabolism and enhanced functioning.
  • ROS are useful as signaling molecules due to their small size and short range, but on the other hand they cause cellular damage if produced in an uncontrolled manner.
  • ROS reactive oxygen species
  • the body does not identify the need to generate ROS.
  • an artificial and controlled source of ROS is required for the desired signaling to the targeted cells.
  • artificially generated ROS at concentrations below that required for cytotoxicity act similarly to enzymatic generated ROS.
  • controlled generation of ROS can have a range of positive effects on the cells and surrounding tissue. Since ROS are involved in many disorders and diseases, treatment methods based on artificially generated ROS can cure, improve, or stabilize diseases or disorders in millions of patients. Indeed, several methods have been suggested to generate beneficial concentrations of ROS in- vivo, using various energy sources, including light, ultrasound and electrical currents.
  • Controlled generation of ROS in vivo may be conducted using nanoparticles loaded with photosensitizers which generate ROS following exposure to light including IR radiation.
  • topical administration of photosensitizer carrying nanoparticles has been suggested for stimulating fibroblast cells for photonic based cosmetic treatments (see for example Hart et al., in US Patent 7,081,128).
  • optical radiation induced ROS production by certain crystal nanoparticles such as ZnO and TiO2.
  • such nanoparticles can also be used as light induced ROS generation.
  • their generation mechanism is based on dangling chemical bonds which exist or are induced at these nanocrystal surfaces. This means that such nanoparticles generate ROS also in the dark, and thus will induce damage in the body until they are cleared.
  • ROS production is not controlled and varies widely versus cell types, electric field level, and so forth.
  • the present invention overcomes these drawbacks of the background art by providing electromagnetic radiation absorbing nanoparticles operable for generating excited species when exposed to electromagnetic radiation in the microwave range. According to other embodiments of the present invention, there are provided methods for generating microbubbles near the nanoparticles by interaction of the excited species induced nucleation with ultrasound. According to still other embodiments of the present invention, there are provided methods and apparatus which employ such nanoparticles for treatments based on controlled generation of excited species.
  • electromagnetic radiation or EMR is defined as radiation having an electric field and a magnetic field propagating at right angles to one another.
  • microwave radiation or MR is electromagnetic radiation selected from the group consisting of terahertz radiation, millimeter waves, microwaves and Very High Frequency radio-frequency radiation.
  • optical radiation is electromagnetic radiation selected from the group consisting of far infra-red, near infra-red, visible, and ultra violet radiation.
  • electromagnettic field or EMF is defined as the Root mean squared (RMS) electric field component of the EMR.
  • absorbing nanoparticle refers to either to single nanoparticles, or . nanoparticles assembled as clusters or agglomerates, exhibiting enhance absorption of specific portion of the electromagnetic spectrum compared to randomly shaped nanoparticles of the same size.
  • ROS reactive oxygen species
  • reactive oxygen species includes but is not limited to Ozone; Hydrogen Peroxide; Hydroxyl Radical; Super oxide anion; Singlet Oxygen; Perhydroxy Radical; Hydroxyl Ion; Hydroperoxy Radical and also nitrogen-oxygen molecules and excited species such as NO, NOOH and other nitrogen oxygen derivatives.
  • NO site or “nucleation bubble” stands for a volume with a finite size measured in nanometers, which upon exposure to suitable ultrasound radiation can be temporarily stabilized or evolved into a larger bubble.
  • thermal nucleation refers to a process during which an absorbing nanoparticle immersed in a liquid is exposed to suitable electromagnetic radiation, heats up above the local boiling point of the liquid and in turn vaporizes a sufficient volume of the liquid to serve as a nucleation site.
  • non-thermal nucleation refers to a process during which an absorbing nanoparticle immersed in a liquid is exposed to suitable electromagnetic radiation, and in turn generates a nucleation bubble, either by generation of non- condensable gas molecules or collecting non-condensable gas molecules from the liquid sufficiently to serve as a nucleation site.
  • cluster is defined as a plurality of nanoparticles spread on a surface of a tissue whose size is measured in a few microns.
  • agglomerate is defined as a plurality of nanoparticles agglomerated in a 3-dimensional structure.
  • P.sub.rjmax is the peak negative (rarefaction) ultrasound pressure in MPa and f the frequency in MHz.
  • FIG. 1 illustrates an exemplary, illustrative but preferred treatment embodiment of skin wrinkles according to the present invention
  • FIG. 2 describes one exemplary, illustrative but preferred embodiment of a method for enhanced transport of materials through the BNB (blood nerve barrier); and
  • FIG. 3 shows an exemplary, illustrative but preferred embodiment of drug release methods from a drug carrying stent utilizing extracorporeal energy sources, according to the present invention.
  • the present invention is of electromagnetic radiation absorbing nanoparticles operable for generating excited species when exposed to electromagnetic radiation in the microwave range. According to other embodiments of the present invention, there are provided methods for generating microbubbles near the nanoparticles by interaction of the excited species induced nucleation with ultrasound. According to still other embodiments of the present invention, there are provided methods and apparatus which employ such nanoparticles for treatments based on controlled generation of excited species. Without wishing to be limited in any way, various applications of the excited species are preferably encompassed within the scope of the present invention, including but not limited to medical applications.
  • Section 1 describes reactive species in liquid environment and methods of generation thereof.
  • Section 2 describes various applications of reactive species for cosmetic treatments.
  • Section 3 describes various applications of ROS for traumatized tissue following ischemia.
  • Section 4 describes various applications of ROS for revitalization of traumatized nerves.
  • Section 5 describes various applications for fluid transport using microbubbles.
  • Section 6 describes applications for water treatment using discharge product generated within microbubbles.
  • Section 1 - ROS, materials thereof and Methods of Generation Thereof there is provided a method for using absorbing nanoparticles for generating ROS in liquid through a non-thermal process, by exposure of the absorbing nanoparticles to EM (electromagnetic radiation).
  • the non thermal process may be characterized by heating which remains below the localized boiling point of liquid at the nucleation site.
  • the nanoparticle is minimally heated during ROS generation.
  • the nanoparticle is in a liquid environment, which preferably includes anything in the liquid local to the nanoparticle but which does not include the nanoparticle itself.
  • Each nanoparticle optionally and preferably behaves as a complete unit.
  • a part of the nanoparticle may optionally behave as a complete unit as for the coating for example.
  • complete unit it is meant that the materials which comprise the nanoparticle do not respond as individual molecules for interacting with EM 5 but rather behave as a cohesive whole
  • the EM preferably comprises MR or light energy, in which light energy is defined as including the infrared (near and far) and visible spectrum of light.
  • MR is preferably used according to some embodiments of the present invention.
  • Magnetic Field Process is a well-known method for overcoming the above problem and for separating water molecules, which are usually tied closely together in clusters, into single individual molecules of water (known as mono-molecules).
  • Fukui et al. in US Patent application 20060263441, teaches that separating the water clusters increases their free energy and thus reduces the energy required to break the free water molecules into free radicals.
  • Zelatova et al. [Ia] claim that a magnetic field can transform the excited water and any dissolved molecules from the singlet state to the triplet state; in the latter state, they can break more easily into two separate radicals which in turn migrate freely. The magnetic field effects may result from the magnetic component of the electromagnetic radiation, or applied externally.
  • Walczak et al. [ 2a] have exposed semiconductor etching solutions to MR in order to conduct efficient reactive ion etching of semiconductor materials. They observed an enhanced reactive etching rate and enhanced directional etching using MR excited etching solution (ie a solution excited by MR). They attribute their observations to changes in the water structure following MR exposure, called a
  • MR exposed solution includes a large amount of free water molecules which in turn enable larger amount of free radicals during etching.
  • the enhanced concentration of radicals increases the etching rate and also the etching directionality similarly to other enhanced etching processes.
  • Another key aspect of MR interaction with aqueous solutions has been reported by Kiel et al., [3a]. They exposed a test tube containing NaHCO3 solution doped with diazoluminomelanin (DALM) to 2-MW (estimated -50 kW/cm2) 5- microsecond 1.25-GHz 1-Hz pulses (0.25 W/cm2 average power). During the pulses the authors observed a strong glow in the test tube and sometimes even localized thin streamer discharges. Clearly, these phenomena (bubble formation, glow, and streamers) were not observed after exposure of the same aqueous solution to a continuous low power MR.
  • DALM diazoluminomelanin
  • the ROS production rate s.subO was measured using the spin trap technique [7a].
  • the nanotubes Prior to MR exposure, the nanotubes were mixed with water solution comprising 5-(diethoxyphosphoryl)-5-methyl-l- pyrroline-iV-oxide (DEPMPO) from Calbiochem. This molecule interacts with various ROS types, yielding relatively stable (half-life of 17.7 min.) species such as W
  • DEPMPO-OOH with superoxide anion and DEPMPO-OH with hydroxyl radical Each generated DEPMPO-x molecule preserves the interacting ROS spin and thus records (i.e., spin-traps) a fraction of the ROS amount generated by the nanotubes during the exposure.
  • the DEPMPO-x spin spectrum is sampled using an Electron Paramagnetic Resonance (EPR) instrument as described by Lavie et al. [7b].
  • EPR Electron Paramagnetic Resonance
  • the spin spectrum was also sampled for material from a vial with a CNT suspension before MR exposure and also from a vial that was exposed to MR but which did not feature the CNT suspension.
  • the generated ROS concentration was calculated by integrating the spectrum after subtracting the base level as described previously [7a].
  • the nanotubes used by Rojas Chapana apparently generated about 60,000 ROS molecules during the exposure period. It is predicted that such amount of generated excited species would be sufficient for breaking of 10,000 chemical bonds in the bacteria membrane, as estimated above to be sufficient for drilling a hole through the bacteria membrane, as observed by Rojas-Chapana.
  • the nanoparticles are first provided to an object to be treated, for example by being administered to and/or applied to the object, in which the object is in a liquid environment.
  • the object is optionally part of a larger object, for example optionally and preferably comprising a portion of tissue to be treated in a body of a subject.
  • the nanoparticles are then exposed to suitable MR, and a plurality of ROS is generated at close proximity to the nanoparticle(s), which is then released into the proximal liquid environment.
  • the MR frequency is between 20 MHz and 1000 GHz, and more preferably between 100 MHz and 3 GHz.
  • the electromagnetic source pulse width may optionally and preferably vary between 10 nanosecond and 30 milliseconds and more preferably between 0.01 and 10 microsecond.
  • the peak MR power density optionally and preferably ranges from about 0.1 kW/cm2 to about 1 MW/cm2, and more preferably from about 1 kW/cm2 to about 100 kW/cm2.
  • the microwave source for treating a subject according to the present invention is optionally and preferably so arranged so as to efficiently couple the microwave energy into the region of interest.
  • the coupling is optionally and preferably attained by an array of microwave sources such as those described by Xiang et al in US Patent application 20070168001.
  • the electromagnetic radiation source is optionally and preferably coupled to the specific patient region by a waveguide equipped with a matching terminating emitter, a metal wire structure, or a compact RF applicator coupled to a catheter.
  • the MR may optionally and preferably be employed in a mode selected from a group of single pulse mode, pulse train mode, repeated sequence mode, or any other time sequence suitable for generating ROS near the absorbing nanoparticles.
  • the absorbing nanoparticles immersed in liquid and are exposed to MR in the presence of magnetic field may be constant, pulsed, alternating, or modulated in any other time sequence. Its intensity frequency may range between 1 Hz to 1 MHz. Its peak field may range between
  • the magnetic field modulation may be in synchronization with the MR intensity sequence.
  • Nanoparticle Materials According to preferred embodiments of the present invention, certain types of nanoparticles are preferred for effective generation of ROS through the application of suitable MR.
  • nanoparticles are operable for enhanced absorption or enhanced scattering of MR as single nanoparticles or as clusters.
  • the nanoparticles interaction cross section at MR frequency may vary from about 0.05 to about 0.5 of their geometric cross section.
  • the peak local EM adjacent to an exposed nanoparticle is at least 5 times the ambient EMF and is referred to herein as "enhanced", such that preferably the nanoparticles generate enhanced localized EMF.
  • the size of the absorbing nanoparticles is preferably selected to be suitable for the method of use. Suitable shapes of absorbing nanoparticles preferably include but are not limited to various types of nanotubes, high aspect ratio rods or ellipsoids, etc.
  • a non-symmetrical nanoshell construction for absorbing nanoparticles optionally and preferably comprises a metal shell and silica core whose center does not coincide with the gold shell center [described by Wang et al. [8a] .
  • the local EMF near such absorbing nanoparticles may enhance the ambient EMF by a factor of up to 60 for THz EMR.
  • the absorbing nanoparticles may optionally comprise local nanometer sized structures which enhance the local EMF in their vicinity.
  • nanotubes, and especially multiwalled nanotubes comprise nanometer sized structures at their tips, which in turn, exhibit EMF enhancement factor up to 2 higher from the value predicted for simple rod shaped absorbing nanoparticles.
  • the absorbing nanoparticles comprise any metal, metal alloy, combinations of metals and non-metals, and non-metals.
  • the nanoparticles may optionally comprise a single material, such as gold or carbon, or can be layered structures, such as silica shapes covered with gold shells.
  • at least one of the nanoparticles structure materials is conductive at MR frequencies.
  • the layered nanoparticles optionally include the asymmetric nanoshells configuration described by Wang et. al., [8a].
  • the absorbing nanoparticles optionally include a metal core with a large aspect ratio, within a jacket preferably made of a glass-like composition.
  • the absorbing nanoparticles are optionally fabricated by microwire drawing technologies, i.e, similar to fiber optic drawing process, with metal wire fed from the center of the drawing nozzle.
  • the metal core diameter of the absorbing nanoparticles optionally ranges from about 10 nm to about 1000 nm.
  • the absorbing nanoparticles optionally have a nanotube shape. They are conductive, and preferably multiwalled. Their diameter preferably varies from about 3 to about 20 nm and their longer dimension preferably varies from about 20 to about 2000 nm.
  • the type of nanotubes is optionally and preferably selected from the group consisting of carbon nanotubes (CNT), Boron nitride nanotubes, BCN nanotubes, in which some carbon atoms were replaced by nitrogen and boron atoms (BCNT) silicone carbide nanotubes, bundles of single-wall carbon nanotubes, multi-wall carbon nanotubes, buckyrubes, fullerene tubes, carbon fibrils, carbon nanotubules, carbon nanof ⁇ bers, and combination thereof.
  • the multi walled nanotubes preferably comprise multiple "walls" in their structural composition, according to the construction material. They may be in the shape of nanoscrolls, nanofibrils, nanovessels, nanocontainers, and combinations thereof. They may comprise a variety of lengths, diameters, chiralities, number of walls, and they may be either open or capped at their ends (for example see Bianco et al. in US patent application 20060199770).
  • the absorbing nanoparticle preferably includes at least one site comprising compositions and/or structures which promote the accumulation of gas molecules generated by its exposure to MR.
  • the site comprises a discontinuity suitable for accumulation of gas molecules as an attached nanobubble.
  • the surface of the absorbing nanoparticle preferably comprises at least one site suitable for accumulation of non-condensable gas molecules in a nucleation bubble, whose surface morphology selected from a group comprising: a crack, a depression, a linear edge, a pointed edge, a boundary between hydrophobic and hydrophilic materials, and a boundary between different surface characteristics.
  • the nanoparticles are used for biological functions, for example for treatment of a subject or some cases for biological treatment outside of a subject.
  • one or more materials for biological functionalization of the nanoparticles are employed as described herein.
  • the absorbing nanoparticles are preferably coated with one or more materials which preferably provides at least one of the following functions: enable efficient ROS generation and inducing nucleation sites, when immersed nanoparticles are exposed to MR, stabilize the absorbing nanoparticles in liquid to prevent their aggregation; minimize uptake of absorbing nanoparticles by the immune system if administered to a body of a subject; serve as an intermediate layer for attachment of targeting ligands; target the nanoparticles to specific cells, tissue or non-tissue materials, enhance nanoparticle transport through blood vessels and interstitial regions naturally or using an external energy source;. Clear the circulation after completion of desired biological use.
  • the coating may optionally contain one or more of a surfactant, linker, spacer, targeting ligand and encasing ligand, as will be explained below.
  • the size of the absorbing nanoparticles is crucial in order to fulfill the above biological functions.
  • the size is more preferably affected from considerations including but not limited to a characteristic selected from the group consisting of the bio-distribution, penetration through vasculature and interstitial volume, and the blood clearance rate, or a combination thereof.
  • the single nanoparticle size could range from about 10 to about 1000 nm.
  • Nanoparticles transport considerations favor the use of absorbing nanoparticles with a smaller dimension range between 10 and 100 nm. .
  • An optimal absorbing nanoparticles size will be determined depending upon their shape, structure, materials, operation conditions and the details of the specific biological application.
  • the present invention in some embodiments, encompasses the use of absorbing nanoparticles comprising both coatings that are covalently bound to the surface of the absorbing nanoparticles and coatings that physically adhere to the surface of the absorbing particle.
  • the latter method of binding will generally be more preferred.
  • the coating material may comprise any of the elements. However, coatings comprising carbon, oxygen, nitrogen, hydrogen, sulfur and phosphorous are preferred.
  • the absorbing nanoparticles are coated with materials which ensure adequate functionalization while preserving the generation of ROS when immersed in liquid and exposed to suitable MR. More particulary, the coating may be conformal and leave uncovered or partially covered sections on the absorbing nanoparticles surface, preferably in regions of EMF enhancement, near which ROS are typically generated. In other aspects the coating material and process minimally interfere with the hydrophobic properties of the gas accumulation site. In yet other aspects, the nanoparticles includes at least two surfaces with distinct characteristics such as gold and silica [2Oa]. Sufficient functionalization is attained by fixating suitable chemical groups to one surface while the other surface is left uncoated and operable for ROS generation.
  • the absorbing nanoparticles are preferably coated with amphiphilic material such as one or more surfactants for example, so as to stabilize their suspension in aqueous solution and to prevent their spontaneous aggregation without rendering their ability to generate nucleation site.
  • the surfactants may comprise one or more of polymeric, block copolymer, or non- polymeric surfactants, preferably to stabilize the absorbing nanoparticles.
  • the surfactants are proteins, modified proteins or other biological molecules as surfactants. Particularly desirable surfactants are materials which enable ROS generation adjacent to the nanoparticles due to exposure to MR.
  • the coating preferably extends the circulation lifetime of the nanoparticles by minimizing uptake by the immune system when administered to a body of a subject.
  • nanoparticles are taken up by macrophages in the body or the RES (reticuloendothelial system), and are hence cleared by the immune system.
  • the surfactant preferably serves as a platform for the attachment of other chemical species with desirable biological or chemical properties, such as targeting ligands.
  • a coating material comprising reactive functional groups is desirable.
  • Reactive functional groups suitable for the present invention include, but are not limited to hydroxyl groups, thiol groups, amine groups, hydroxyl, halo, cyano groups, carboxyl, and carbonyl groups, as well as carbohydrate groups etc.
  • the attached species such as targeting ligands and the coating comprise one or more reactive functional groups.
  • the attachment process may be optionally performed using a linker (an endured connection) and a spacer (cleavable attachment). Both linker and spacer preferably have a pair of reactive functional groups.
  • the spacers may optionally comprise linker groups that are cleavable under the action of enzymes, acids, bases, and other chemical or biological entities. With such cleavable spacers, the absorbing nanoparticles may modify their properties over time. For example, before cleavage of the spacer, the nanoparticles may optionally have a strong affinity for certain cells, organs, or non-tissue material. After cleavage, they are preferably rapidly cleared from the body.
  • the nanoparticles comprise surfactants with modifiers attached directly or through linkers and spacers that are cleavable under the action of enzymes, bases, acids, or other chemical entities.
  • the linker or spacer species are attached to the nanoparticle coating.
  • the invention encompasses the use of absorbing nanoparticles to which one or more targeting ligands are attached, either through a chemical bond or through direct interaction with the nanoparticle surface.
  • These targeting ligands are optionally operable to promote absorbing nanoparticles accumulation within the targeted biofilm.
  • the targeting ligands may optionally be attached directly to the surface of the absorbing nanoparticle or attached indirectly through the surfactant.
  • the targeting ligands may optionally comprise any chemical group that binds to a "targeting receptor" associated with the targeted cells, tissue or non-tissue material.
  • the targeting ligands can be derived from any synthetic, semi-synthetic, or naturally occurring chemical species. Materials or substances that can optionally serve as targeting ligands include but are not limited to amino acids, peptides, proteins, antibodies, antibody fragments, hormones, glycoproteins, lectins, sugars, saccharides, carbohydrates, vitamins, steroids, hormones, cofactors, and genetic material, including nucleosides, and nucleotides, and the like.
  • the targeting ligand can optionally be either an independent molecule or a molecular fragment.
  • the molecular weight of the targeting ligands is preferably selected to provide adequate attachment fraction of the nanoparticles to the target cells, tissue or non- tissue materials.
  • a typical ligand molecular weight suffient for attachment onto a cell should be at least 10 kd (kilo Dalton) to avoid detachment during the application of ultrasound energy.
  • the receptors for which the targeting ligands have a special affinity are preferably chemical groups, proteins, or other species that are overexpressed and/or specifically expression by the targeted biofilm, or bacteria membrane. In general, terms the receptors can be any chemical feature of the targeted biofilm or bacteria.
  • the receptors can also optionally be independent chemical entities in the blood or other body fluids, including externally administered drugs, drug components, or drug metabolites.
  • the absorbing nanoparticles are preferably coated with a lipid composition for enhanced extravasation, tissue penetration, and enhanced circulation lifetime.
  • the nanoparticles are entrapped in liposome.
  • the lipid coating enables ROS generation adjacent to the absorbing nanoparticles.
  • the liposome optionally and preferably comprises one or more targeting ligands.
  • the absorbing nanoparticle maintains ability to generate nucleation site and its evolution, possibly into a microbubble, while in the liposome.
  • the smaller dimension of the nanoparticle with the lipid coating ranges between 20 and 200 run.
  • Solid nanoparticles suitable for the present invention are preferably robust and in turn are typically functionalized by attachment of amphophilic material as described above to their surface, through chemical reactions.
  • the functionalization process for nanotubes is more complicated because of their delicate structure and inertness of their basal structure envelope.
  • certain functional molecules may be attached to the nanotube tips while their envelope may be uncoated.
  • Bianco et al. in US patent 20060199770 describe various methods for CNT functionalization and targeting ligands attachment, mainly onto CNT tips.
  • the functionalization process and materials are preferably selected and optimized to leave the tips with minimal coating, enabling ROS generation and yet to ensure adequate functionalization.
  • a non-thermal method for generating a nucleation site near an absorbing nanoparticle in a liquid environment to an EMF without wishing to be limited by a single hypothesis, it is believed that the formation of sufficient amount of ROS near absorbing nanoparticles can generate a nucleation site.
  • the term "non-thermal nucleation” means that the peak liquid immersed nanoparticle temperature during non-thermal generation of the nucleation site does not exceed the boiling point of the liquid at the pressure within the nucleation site.
  • These ROS preferably cause gas molecules to be generated in close proximity to the absorbing nanoparticle.
  • non-condensable gas molecules more preferably accumulate on the nanoparticle(s) to form a nucleation site.
  • the interaction of absorbing nanoparticles with the MR induces the accumulation of dissolved gas molecules on the nanoparticle(s).
  • the generation of non-condensable gas molecules may optionally result from disintegration of a molecule with favorable path for its generation, such as NaHCO3 and by bond breaking in larger organic molecules.
  • the non-condensable gas molecules include but are not limited to one or more of C02, 02, CH4, NH3, etc.
  • liquid preferably includes one or more of water, aqueous solution, non-aqueous solution, gel, semi-solid, suspension, dispersion, membrane, liquid comprising a solid matrix, etc. These molecules may be dissolved, suspended, emulsified, part of a gel matrix and so forth.
  • the size of the nucleation bubble theoretically depends on the number of contained non-condensable molecules and also on the internal pressure which is the sum of Laplace pressure and the ambient pressure as described in Eqn. (4)
  • R s P ⁇ and ⁇ st are the nanobubble radius, ambient pressure and the liquid surface tension.
  • R s P ⁇ and ⁇ st are the nanobubble radius, ambient pressure and the liquid surface tension.
  • 400,000 non-condensable molecules form a nucleation bubble whose radius is 50 nm, having an internal pressure reaches 2.9 MPa.
  • one possible threshold estimate is of order 100,000 gas molecules with radius of 25 nm.
  • Attard et al. [9a] have found that hydrophobic regions on surfaces tend to accumulate spontaneously and retain gas molecules as attached to surface nanobubbles. They further found that the internal pressure inside these surface nanobubbles is much lower than would be expected according to the Laplace pressure.
  • Agrawal et. al., [10a] characterized the spontaneous accumulation of gas nanobubbles on hydrophobic patches. They found that a large number of nanobubbles accumulate spontaneously on submicron hydrophobic patches and that the thickness of the nanobubble layer is a few tens of nanometers. From these findings, it is estimated that nanobubbles comprising between 10,000 and 100,000 molecules can survive on hydrophobic surfaces.
  • the generated non-condensable gas molecules accumulate on the absorbing nanoparticle due to mechanisms such as reducing the free surface energy.
  • the non-condensable gas molecules are accumulated on accumulation promoting site(s) on the absorbing nanoparticle surface resulting in the formation of a nucleation bubble.
  • P. sub B is the Blake pressure which should be equal to the ultrasound rarefaction pressure in order to grow a nucleation bubble.
  • Farny et al showed growth for thermal nucleation bubbles whose Blake radius and pressure are 80 nm and 1 MPa.
  • nanobubbles accumulated on hydrophobic surfaces which may serve as nucleation sites, especially since they are less pressurized, and thus are expected to respond to lower ultrasound rarefaction pressures.
  • recent experiments [12a] demonstrated that flat nanobubbles whose equivalent volume is equal to a Blake radius sphere, on a flat, uniform and clean surface, are stable and do not serve as nucleation sites, even under strong (6 MPa) ultrasound insonation.
  • the present invention provides methods for generation of the nucleation site through exposure of absorbing nanoparticles to MR, thereby inducing intensive accumulation of non-condensable gas. It is further predicted that such MR induced accumulation in suitable accumulation sites may increase the nanobubble size beyond the size of spontaneously forming nanobubbles, and thus enable generation of nucleation bubble and at lower ultrasound peak rarefaction pressures.
  • the nucleation bubble is induced by MR "extraction" of gas molecules from the liquid near the absorbing nanoparticle.
  • Generation of microbubbles from a cluster of absorbing nanoparticles may optionally be performed as follows.
  • WO 2006 051542 by the present inventor taught a method for efficient generation of microbubbles from thermal nucleation bubbles around electromagnetic radiation absorbing nanoparticles, which are exposed to electromagnetic radiation and in particular light.
  • the present invention may use ultrasound radiation in combination, for preventing the nucleation bubble from redissolving into the liquid and for causing it instead to grow through rectified diffusion. Rectified diffusion occurs when ultrasound energy causes supersaturated gas to be pumped into an existing small nanobubble, making the bubble increase in size.
  • WO 2006 051542 teaches a method for generating a microbubble from a cluster of nucleation bubbles using the coalescence effect, explained in Krasovitzki et al. [19a]. Exposing a cluster of absorbing nanoparticles to ultrasound energy can significantly reduce the ultrasound intensity required for generating a microbubble.
  • the absorbing nanoparticles are fabricated so as to enhance their attachment onto target objects as clusters.
  • the present invention provides methods for enhancing ROS flux uniformity on target cells membranes using microbubbles, evolving by ultrasound.
  • the evolved microbubbles induce relative motion of the absorbing nanoparticles in respect to the adjacent targeted cells so as to spread the ROS generation around the membranes.
  • the useful ROS concentration in the interstitial fluid must be in the range of 10.supl3 to 10.supl5 ROS/cm.sup3 in order to induce cellular stimulation [14a ].
  • concentrations require absorbing nanoparticles require the density to be on the order of 10.sup.9 /cm.sup.3 or less.
  • the inter-nanoparticle distance would be iO micron or roughly one nanoparticle per cell.
  • the range of ROS may be a few hundred run from the generating nanoparticle. Therefore, such localized generation of ROS would result in cellular damage rather than the desired signaling effect.
  • microbubbles generated near the absorbing nanoparticles induce microstreams (see for example Longuet-Higgins [15a]).). These microstreams, whose speed may exceed 1 cm/sec, translate the interstitial nanoparticles in respect to the adjacent cell(s). In turn, a relatively uniform flux of ROS is incident on the targeted cell(s) membrane, inducing the desired signaling effect, while minimizing the damage probability.
  • the present invention provides methods for inducing a relatively uniform flux of ROS around target cell(s) surface comprising the stages of: a) administering one or more active absorbing nanoparticles to a targeted cell; (b) positioning an ultrasound source and a source of MR so as to couple their energy to the cell(s); (c) exposing the cell(s) to simultaneously MR ultrasound energy so as to generate ROS at close proximity to the nanoparticle(s); (d) further exposing the nanoparticle(s) to simultaneous MR and ultrasound radiation so as to generate a nanobubble near a fraction nanoparticle(s) and evolve the nanobubble into a microbubble; and (e) further exposing the nanoparticle(s) to the ultrasound radiation so as to pulsate the microbubble and translate at least a fraction of the nanoparticle(s) in respect to the cell(s) adjacent to it, resulting in relatively uniform flux of ROS onto the cell(s).
  • Microbubbles induced ROS generation
  • Bertuglia [18a] exposed cardiac tissue to periodic pulses of moderate ultrasound post ischemia. Bertuglia found that ultrasound induced microbubbles pulsation in close proximity to the tissue results in increase of endothelial NO production during post ischemic reperfusion. The amount of generated NO during short time exposure was sufficient to enhance microvessel growth.
  • the present invention provides methods for inducing ROS in target cell(s) comprising the stages of: a) administering one or more absorbing nanoparticles to a targeted cell; (b) positioning an ultrasound source and a source of MR so as to couple their energy to the cell(s); (c) exposing the cell(s) to MR so as to generate ROS at close proximity to the nanoparticle(s); (d) exposing of the nanoparticle(s) to ultrasound radiation so as to generate at least one nanobubble near at least a fraction of the nanoparticles and evolve the nanobubble into a microbubble; and (e) further exposing the microbubble to the ultrasound radiation so as to pulsate the microbubble and induce ROS generation within the cell(s).
  • the ultrasound energy frequency for evolving a microbubble from a nucleation site varies between about 20 kHz and 10 MHz and more preferably between 0.5 and about 10 MHz.
  • frequency for therapeutic ultrasound preferably ranges between about 0.75 and about 3 MHz, with from about 1 and about 2 MHz being more preferred.
  • energy levels may vary from about 0.5 Watt (W) per square centimeter (cm.sup.2) to about 20 W/cm.sup.2, with energy levels of from about 0.5 to about 2.5 W/cm.sup.2 being preferred.
  • the MR is pulsed or pulse periodic, and the ultrasound peak rarefaction may be synchronized with the MR pulse(s), preferably at a specific location within the region where the generation of nucleation sites is desired.
  • the ultrasound peak rarefaction pressure may optionally be at a phase delay of 1/8 of its cycle in respect to the peak microwave electric field.
  • the ultrasound peak rarefaction delay may optionally and preferably be varied such that the ultrasound peak rarefaction is delayed by about 1/8 of a cycle in respect to the MR pulse at a specific location within the region.
  • the ultrasound energy used for treating a subject according to the present invention may optionally and preferably be introduced to the targeted tissue (generally, superficial tissue) region by positioning external ultrasound source.
  • the targeted tissue generally, superficial tissue
  • deep tissue treatment may require focusing the ultrasonic energy so that it is preferentially directed within a focal zone.
  • sources are HIFU sources composed of an array of ultrasound sub sources.
  • the ultrasonic energy may optionally be applied via interstitial probes, intravascular ultrasound catheters, or endoluminal catheters, typically composed of mechanically insulated metal wire.
  • Silica coated gold rod absorbing nanoparticles were prepared in suitable methods such that the silica does not cover the gold rod tips.
  • the gold core is a rod whose curved radius is 50 nm and dimensions are about 10 nm diameter, 100 nm long while the silica coating diameter is 25 nm.
  • the silica coating is functionalized through standard methods so that its surface is coated with O-Si-(CH.sub.3).sub.3 (silane) groups.
  • the silica region close to the nanoparticles tips is modified to be hydrophobic using a polystyrene like groups attached to the silane groups.
  • a suspension of the nanoparticles whose concentration is 10.sup.9 nanoparticles per cm.sup.3 were mixed with E.
  • nanoparticles for 10 minutes and rinsed with DI water. After rinsing and centrifugation, the nanoparticles accumulated on the E. coli bacteria surface in clusters of 10 — 50 nanoparticles (about 100 nanoparticles per bacteria).
  • a 10 ml 0.6% saline solution comprising the nanoparticles loaded bacteria (3*10.sup.5 bacteria per cm.sup.3) and suitable organic buffer was placed in a square cross section test tube. The test tube was then placed next to a waveguide coupled to a 1 GHz pulsed MR source. A non-conductive rod coupled ultrasound energy from a transducer was placed outside the waveguide and the test tube using suitable gel.
  • the test tube was then exposed to 0.5 microsecond 1 GHz microwave pulses whose intensity at the test tube location, is 5 kW/cm2 at 0.25 Hz rate for a period of 1 minute.
  • the ROS disintegrated the bacteria membrane and generate CO.sub.2 and other non-condensable gas molecules.
  • 100,000 non- condensable gas molecules accumulated near the tips as a nanobubble whose equivalent diameter is 50 nm.
  • the test tube was exposed to 3 MHz ultrasound energy guided through the rod, in such way that the peak overpressure within the test tube was 0.5 MPa. After about 1 minute of simultaneous exposure to microwave pulses and ultrasound, the microbubble population within the test tube reached 10.sup.5 microbubbles per cm.sup.3, as measured by ultrasound reflection from the test tube through the guide rod.
  • Section 2 Applications of absorbing nanoparticles for cosmetic treatments.
  • This Section relates to cosmetic treatments with the methods according to the present invention.
  • a first example relates to stimulation of fibroblast cells for restructuring of collagen, such as for example in the facial skin of a subject.
  • a second example relates to treatments of dermatological conditions in deep skin layers.
  • the present invention provides methods for cosmetic treatments including: hair growth management, including limiting or eliminating hair growth in undesired areas and stimulating hair growth in desired areas, treatments for PFB (Pseudo Follicolitus Barbe), skin rejuvenation, removing cellulite, skin anti- aging including improving skin texture, pore size, elasticity, wrinkles and skin lifting, improved skin moistening, removal of pigmented lesions, repigmentation, tattoo reduction/removal, psoriasis, reduction of body odor, reduction of oiliness, reduction of sweat, reduction/removal of scars, prophylactic and prevention of skin diseases, and for treating and/or preventing disorders of follicles including acne vulgaris.
  • hair growth management including limiting or eliminating hair growth in undesired areas and stimulating hair growth in desired areas
  • treatments for PFB Pseudo Follicolitus Barbe
  • skin rejuvenation including limiting or eliminating hair growth in undesired areas and stimulating hair growth in desired areas
  • skin anti- aging including
  • the present invention further provides in some embodiments treatment methods for dermatological conditions in deep skin layers such as burns, acne, herpes simplex, psoriasis, skin cancer and ulcers including infected or non-infected chronic ulcers of different etiology such as venous ulcers, diabetic ulcers, decubitus ulcers, pressure sores, burns and post-traumatic ulcers.
  • treatment methods for dermatological conditions in deep skin layers such as burns, acne, herpes simplex, psoriasis, skin cancer and ulcers including infected or non-infected chronic ulcers of different etiology such as venous ulcers, diabetic ulcers, decubitus ulcers, pressure sores, burns and post-traumatic ulcers.
  • the present invention provides methods in some embodiments for reconstructing collagen layers using excited species generation from absorbing nanoparticles exposed to MR, for example for treatment of wrinkles.
  • Wrinkles can be roughly divided into fine and medium wrinkles and folding. Due to mechanical stresses, the relatively flat collagen fiber in the dermis follows the shape of the wrinkles and bends, thus fixating the wrinkles. In certain locations, the combination of extensive skin movement and muscles stress results in coalescing several wrinkles into a medium wrinkles and even into a fold where the distorted collagen layers may be as deep as 4 mm.
  • the use of effective cosmetics hydrates the epidermis, reduces its elastic module and in turn reduces the wrinkles amplitude, but can not restore the fixated dermis structures underneath. Therefore, there is a need for effective treatment that would remove wrinkles by restoring the flat structure of the collagen fiber while reducing the dermis elastic coefficient.
  • Photo rejuvenation has been a well established method for skin rejuvenation for more than ten years.
  • Traditional treatments employ laser irradiation to remove wrinkles, while causing minor burn wound to the skin.
  • More recently, properly selected visible wavelengths were proven effective in removing wrinkles.
  • photo rejuvenation is based on two stages: In the first stage, the deformed collagen is thermally damaged, for example by optical power which can be selectively absorbed in the collagen layer [2b]. In the second stage, the photo induced ROS stimulate fibroblast cells within the collagen matrix by signaling. The stimulated fibroblast cells proliferate and use the disintegrated collagen to construct flat and ordered collagen layers.
  • ROS light induced Reactive Oxygen Species
  • ROS light induced Reactive Oxygen Species
  • Lubart [6b] suggested that exposing skin to light doses of visible light at the energy doses of 20-30 J/cm 2 , produces sufficient amount of ROS to destroy old collagen fibers and encourage the formation of new ones. At somewhat lower ROS generation rates, the fibroblasts proliferate, regenerating the skin supporting tissue and improving its visual appearance.
  • Lavie et al. [7b] measured the ROS production levels and Ca+2 intake rate of cardiac cells exposed to accumulated light doses of 3.6 and 12 J/cm2. Apparently, these doses are generated at depth of 0.5 and 2 mm respectively, by application of 30 J/cm2 light to the skin. They found that following exposure to 3.6 J/cm2, the peroxide content increased by 60% while Ca+2 concentration increased gradually by 10% for more than an hour. The estimated ROS concentration within the cells is estimated as lO.supH to 10.supl5 ROS per cm.sup 3. These results seem to explain the performance of modern photo-rejuvenation treatments which appear effective in removing fine and medium wrinkles for a limited period. In such treatment skin is exposed to 30 - 50 J/cm2 in the spectral range of 500 - 600 nm which decays to 10% at depth of 1 - 2 mm.
  • photosensitizer materials for skin rejuvenation.
  • controlled amounts of photosensitizers are administered to the dermis of damaged skin and exposed to red light for ROS generation. Since photosensitizers do not penetrate to fibroblast membrane, the fibroblast cells during such treatment are probably stimulated by ROS acting in the signaling mode.
  • photosensitizer molecules such as ALA, when carried by nanoparticles and excited by NIR radiation in the spectral range of 700 — 800, were found to generate ROS.
  • NIR radiation in the range can penetrate at least 5 mm in many skin types, a depth suitable for treating folding lines.
  • photosensitizer generated ROS may be utilized both for collagen destruction and fibroblast stimulation.
  • the ROS generation depth profile resulting from photosensitizer penetration profile and the exponential decay of the light intensity renders this treatment unsuitable for deep layers of skin.
  • treatment parameters suitable for superficial fine wrinkles would not stimulate deep fibroblast cells, while deep folding line treatments may be ineffective for treatment of fine and medium wrinkles.
  • the nanoparticles are introduced to the dermis using ultrasound energy operated using suitable parameters.
  • the nanoparticles are applied on the skin following suitable level of microdermal abrasion.
  • the nanoparticles are introduced by ionophoresis processes, operated with an integrated electrical source or by using a suitable electrical handle following the application of topical gel.
  • the present invention provides methods for cosmetic treatments in a skin region comprising the stages of: a) administering adequate concentations of absorbing nanoparticles to fibroblast cells within target skin; (b) positioning an ultrasound source and a source of MR so as to couple their energy to the skin region; and (c) exposing the desired skin region to MR so as to generate ROS adjacent to the nanoparticles.
  • the stimulated fibroblasts consume the disintegrated collagen and rebuild flat collagen layers which in turn promote skin rejuvenation.
  • Fig. 1 shows an exemplary, illustrative but preferred treatment embodiment of skin wrinkles according to the present invention.
  • Fig. IA shows a detailed cross section of skin region 100 comprising a wrinkle 108 visible outside the epidermis 105. Beneath wrinkle 108 there is a distorted dermis layer 110 which includes distorted surfaces of collagen 150, fibroblast cells 112 and matrix tissue 115.
  • the structure of skin region 100 is preferably treated through the administration of absorbing nanoparticles 125, preferably with targeting ligands, operable for attachment to the fibroblast cells 112, preferably through topical administering.
  • An extracorporeal energy source which is preferably a MR (microwave radiation) source 130 as shown, is preferably positioned adjacent to the skin region 100 and operable for emitting MR 135, optionally periodically or intermittently.
  • the dermis layer 110 is heated, so as to convert at least a fraction of the collagen surface 150, into a disintegrated collagen surface 160.
  • the heating is preferably conducted through the MR source 130 alone or with other energy sources (not shown), with suitable parameters for disintegrating of collagen layers 130.
  • the administered nanoparticles 125 diffused into the dermis layer 110, naturally or using ultrasound energy 145.
  • the nanoparticles 125 During diffusion, a significant fraction of the nanoparticles 125 attach to the fibroblast cells 112. The nanoparticles 125 generate ROS 155 following exposure to MR 135. The fibroblast cell 112 is stimulated by the ROS 155 generated at close proximity to them, thus enhancing its metabolism for an extended period.
  • FIG. 1C A detailed view of the cellular level process induced as a result of the skin wrinkle treatment is shown in Fig. 1C.
  • the enhanced metabolism in fibroblast cell 112 leads to consumption of the disintegrated collagen 160 at an enhanced rate, as well as to more rapid rebuilding of a nearly flat collagen surface 165.
  • the stimulation process described above is repeated until all the disintegrated collagen surface 160 is rebuilt into a new and relatively flat collagen surface 165.
  • the elastic module of the dermis layer 110 within the skin region 100 is modified, resulting in modified stresses in the layers within the skin region 100, thus eventually eliminating the wrinkle 108.
  • a suitable ultrasound source 140 is also positioned to expose the disintegrated collagen surface 160 and fibroblast cell(s) 112 to ultrasound energy 145.
  • the simultaneous exposure of nanoparticles 125 to MR 135 and ultrasound energy 145 with suitable operating parameters induces at least one nucleation site adjacent to the fibroblast cell 112 which evolves under suitable operation parameters of the ultrasound energy 145 into a microbubble 158 adjacent to the fibroblast cell 112.
  • a directional liquid motion is induced in the interstitium adjacent to the fibroblast cell 112 and migrate the nanoparticles 125 from their initial locations.
  • nanoparticles 128, optionally including one or more nanoparticles 125 are now adjacent to fibroblast cell 112.
  • the nanoparticles 128 also generate a pulsating microbubble 170 through the process described above and may also migrate the nanoparticles 128 in respect to the fibroblast cell 112.
  • the ROS 155 are generated in various locations on the fibroblast cell 112 membrane. Accordingly, the fibroblast cell 112 membrane surface is exposed to a relatively uniform incident flux of ROS 155, thus minimizing possible induced damage to fibroblast cells 112.
  • a method for inducing ROS 155 within a fibroblast cell 112 is shown in Figure IE.
  • a suitable ultrasound source 140 is also positioned to expose the disintegrated collagen surface 160 and fibroblast cell(s) 112 to ultrasound energy 145.
  • the simultaneous exposure of nanoparticles 125 to MR 135 and ultrasound energy 145 with suitable operating parameters induces at least one nucleation site adjacent to the fibroblast cell 112 which evolves under suitable operation parameters of the ultrasound energy 145 into a microbubble 170 adjacent to the fibroblast cell 112.
  • the microbubble 170 pulsates in such amplitude as to induce ROS 155 generation within adjacent fibroblast cells 112.
  • the combined fibroblast cells 112 stimulation from adjacent and internal ROS 155 generation a may further enhance the consumption of disintegrated collagen surface 155. This effect complements and enhances the wrinkle 108 elimination level following the multiple treatment sessions.
  • the ultrasound energy frequency for evolving nucleation sites into microbubbles varies between about 300 kHz and 10 MHz and more preferably between 0.5 and about 5 MHz.
  • the average ultrasound energy levels may vary from about 0.5 Watt (W) per square centimeter (cm.sup.2) to about 20
  • the nanoparticle depth profile is controlled so as to induce relatively uniform collagen reconstruction throughout the dermis layer thickness.
  • a uniform collagen rebuilding may remove even medium wrinkles and folding resulting with effective skin rejuvenation.
  • the nanoparticle properties and the ultrasound operating parameters are used to tailor the nanoparticle depth profile suitable for the wrinkles and folding at the specific treatment region.
  • at least a portion of the absorbing nanoparticles penetrate the target cells, and generate in the cells ROS which affect intercellular component such the mitochondria. Phototreatments based on this mode of operation mimic some of the phototherapy processes, were described by Lubart et al. [6b].
  • absorbing nanoparticles of the present invention suitable for acting within cells have the proper structure, materials, coating and ligands for minimizing damage to the target cells following penetration.
  • the upper dermis temperature within the wrinkle region is increased using a heat source so as to obtain the optimal collagen production.
  • a non-contact mid-infrared thermal sensor could be used to monitor dermal temperature.
  • the raw materials are externally supplied to the treated skin after the treatment to support enhanced fibroblast cell activity.
  • a process in accordance with the present invention may be used to provide short or long-term control, improvement, reduction or elimination of acne or other related skin diseases.
  • the skin cells are unable to handle properly bacterial and other contamination in localized region.
  • the acne bacteria attach to the sebaceous glands and start to multiply in various locations on their wall.
  • the hair shaft becomes inflamed and swells from toxins released due to the bacterial activity, which also induce redness in the skin around it.
  • the skin appearance becomes spotted with red flaws, with certain social and cosmetic implications.
  • the absorbing nanoparticles of the present invention are administered to cells of the sebaceous (oil) glands, ducts, or supporting tissue. Exposure of the nanoparticles to MR generates ROS, stimulate the target cells and promote their attack on the acne bacteria.
  • the acne bacterial colony is eradicated, enabling healing of the acne disorder.
  • the treatment provided by the present invention is advantageous in cases the acne bacteria are located deeper than 1 mm, where suitable visible light does not adequately penetrate. Improvement in skin disorders may be a direct or indirect result of the provided treatment, as may be reduced oiliness of the skin, reduced size or diminished appearance of pores, etc.
  • the present invention is also useful for treating enlarged pores, oily skin, and other disorders where there is no active acne-related disorder in some embodiments.
  • Scarring is commonly seen as a consequence of disorders, diseases, or dysfunctions of the sebaceous apparatus. Scarring may consist of one o,r more of the following: raised hypertrophic scars or fibrosis, depressed atrophic scars, hyperpigmentation, hyperpigmentary redness or telangectasia.
  • the present invention provides treatments for raised or thick or hard hypertrophic scars (which are composed of an excess of collagen) by ROS induced stimulation of production of collagen dissolving enzymes (called Matrix metalloproteinases). These enzymes such as MMP-I (collagenase) cause the scar tissue to be diminished.
  • the treatment can be accomplished by administering absorbing nanoparticles to the scar region alone.
  • the scars can be treated in combination with localized thermal methods, for enhancing MMP-I production as described by McDaniel in US Patent McDaniel 6,676,655. [4b].
  • the wound healing process involves various cells types including leukocytes, and platelets.
  • the leukocytes move along the blood vessel walls, migrate through fissures or gaps and begin to attack dying or dead cells. This begins a process of releasing a fluid that combines with a serous substance being extruded from the wall of the blood vessel. Later this process helps in the reduction of pathogenic microorganisms to develop into the blood stream.
  • Another cell known as a platelet, begins the adhesion process to the walls of the damaged vessel.
  • the fibroblast role in wound healing includes synthesizing various types of collagen fibers bundles and structures which serve as contraction matrix for tissue rebuilding (see for example Kurtz in US patent application 20060241495). During the early healing stages, they generate fibrin fibers simultaneously appear forming a fine mesh and developing a "clot" which pulls the damaged edges of the wound together whether this is an internal tear or external laceration. During wound healing, various types of cells generate various ROS types. For example Hoekstra et al., in US Patent application 20070128296 claims that enhanced healing is possible by reducing the concentrations of these ROS types throughout the wound volume, using certain metal salts. They claim that scavenging certain ROS types are important for enhancing wound healing.
  • Phototherapy in the visible range has been shown to be beneficial in the healing of many types of chronic and acute wounds.
  • Whelan et al., [9b] have found significantly accelerated healing of ischemia, diabetic, and chronic wounds following light exposure. They also found that lights help wounds that are normally very difficult to heal such as diabetic skin ulcers, serious burns and the severe oral sores caused by chemotherapy and radiation.
  • phototherapy is involved in ROS generation within wounds. Without wishing to bound to any specific theory, it is claimed that the phototherapy induce ROS generation within the cells involved in wound healing, and some of these cells type are exposed to naturally generated ROS at concentrations well below the level needed for stimulation. It is further claimed that ROS generation is even lower for subject with certain conditions such as diabetes, and during chemotherapy.
  • the present invention provides methods for enhancing wound healing comprising the stages of: a) administering absorbing nanoparticles to a wound region; (b) positioning a source of MR so as to couple its energy to the wound region; (c) exposing the wound region to simultaneous MR so as to generate ROS within the wound region; and (d) stimulating cells in the wound which play role in the wound healing so as to accelerate wound healing in the wound region.
  • the wound region is further exposed to simultaneous MR and ultrasound radiation so as to generate nanobubbles near the nanoparticles and evolve the nanobubbles into microbubbles.
  • the microbubbles are also used to induce ROS generation within a fraction of the cells in order to assist wound healing in the wound region.
  • the microbubbles are used to translate the nanoparticles in respect to the cells adjacent to them, in order to induce relatively uniform flux of ROS onto the cells membranes, thus minimizing damage to the cells due to localized ROS flux.
  • the absorbing nanoparticles are preferably administered through a topical gel for cosmetic treatments according to the present invention.
  • Their delivery into the target which may be tissues, ducts, or glands, may optionally be enhanced, facilitated or made possible by the use of ultrasound, photonphoresis alone or in combination with alteration of the stratum corneum.
  • the nanoparticle delivery into the target may be also enhanced by the use of enzymes capable of altering the structure, permeability, or other physical characteristics of the stratum corneum.
  • An electrical or magnetic charge may be used to enhance their penetration.
  • Microderm abrasion may also be used to permit greater penetration of the skin, wherein the upper epithelial layers are removed. These layers create a natural barrier to the permeability of the skin and by their removal, penetration of the skin by topical agents is facilitated.
  • the nanoparticles are optionally and preferably introduced into the skin micro-vasculature.
  • the absorbing nanoparticles are preferably fabricated to enhance their accumulation adjacent to the targeted skin cells (e.g., fibroblast), tissue or non-tissue material. Enhanced attachment may be attained for example by optionally using specific ligands, MIP technology or pre-determined nanoparticles structures.
  • the active absorbing nanoparticles preferably accumulate on the target cells, tissue or non-tissue material as clusters to promote microbubbles generation following exposure to ultrasound.
  • the absorbing nanoparticles are preferably designed for quick clearance from the skin tissue.
  • the fibroblast cells consume the disintegrated collagen and generate collagen which could be type I or III.
  • the effective generation and reconstruction period may take more than a week and depends on various factors including the supply of "raw materials" as described above.
  • the treatment is optionally and preferably conducted in several weekly or bi-weekly sessions.
  • Photo aged female patients i.e., females experiencing deep wrinkles due to deformation in deep collagen layer are treated in accordance with the wrinkle removal method of the present invention.
  • Each treatment includes application of a thin layer of lotion on the patient face.
  • the lotion comprises active absorbing nanoparticles described in EXAMPLE 1 and compositions which assist their penetration into the collagen generating tissue.
  • the target region of the patient's skin is subjected to suitable RF heating so as to induce selective heating of the dermis layer within the skin to a level sufficient for disintegrating a significant fraction of its collagen surfaces.
  • the target region is preferably simultaneously exposed to 0.5 microsecond pulsed 1.5 GHz microwave source at an intensity ranging from 2 - 4 kW/cm.sup.2 for 30 pulses and ultrasound source operating at 3 MHz having an averaged intensity ranging between 1 - 2 W/cm.sup.2 incident on the skin region for 3 minutes.
  • a sensor attached to the treatment handle ensures that skin temperature is kept below the threshold of thermal injury.
  • the treatment program includes eight treatment sessions over 12 weeks applied onto the entire face of patient. During the period between sessions, a layer of new collagen is generated beneath the skin in the wrinkles region. After eight treatment sessions, the average reduction in visible deep wrinkles exceeds 60%. In addition to wrinkle reduction, several other significant changes are noted including: reduction in brown liver spots and freckles, improved skin tone and elasticity, and a consistently observed 'creamy" color to skin which is caused by new collagen formation.
  • Section 3 Applications of absorbing nanoparticles for infarcted tissue treatments
  • This Section relates to treatments of infarcted tissue with the methods according to the present invention.
  • a first example relates to stimulation of damaged myocardial cells following myocardial infraction.
  • a second example relates to immune traumatized myocardial cells to the cascade of events following reperfusion.
  • the present invention provides a method (in some embodiments) for improving myocardial tissue functioning including but not limited to: reduction of infarct size, regeneration of cardiomyocytes, myocardial revascularization and stimulated angiogenesis in the myocardium, improving function of diseased myocardium, and also optionally cardiac cell biostimulation and additional post- infarct clinical treatment.
  • Heart disease or heart failure following Myocardial Infarction (MI) is still the major cause of death in the western world.
  • the mature heart muscle (myocardium) cells of mammals are those that reach their last stage of differentiation and, therefore, are considered unable to undergo proliferation (see P. P Rumynastev, Growth and
  • Ad and Oron [Ic] list several techniques used for reduction of myocardial infarct size by reperfusion. These include vascular endothelial growth factor (VEGF), coronary artery bypass grafting, percutaneous transluminal coronary angioplasty etc. However, these methods suffer from inconsistency and various adverse effects and require highly skilled medical experts. Oron and Matkovitz in US Patent 7,051,738 claimed that exposing infarcted myocardium to suitable wavelength (e.g., 804 nm) low level light induces infarct size reduction, regeneration of cardiomyocytes in the infarct, preservation of the structure and activity of mitochondria in cardiomyocytes, and improved function of diseased myocardium. Levy, Shainberg et.
  • suitable wavelength e.g. 804 nm
  • Non invasive optical based methods require light penetration through the chest skin and muscles between the ribs with a total average tissue width of about 3 to 5 cm before reaching the heart muscle. Since the optical power diminishes within living tissue in an exponential manner with respect to depth, the maximal optical power incident on the heart would diminish.
  • Oron and Matkovitz in US Patent 7,051,738, suggested introducing the required light dose onto the myocardium by exposing non-myocardial tissue in the patient back or chest.
  • such invasive surgery may involve high morbidity rates and requires special medical expertise.
  • the present invention provides, in some embodiments, methods for stimulating damaged cells following an ischemia event, thereby overcoming the above drawbacks of the background art.
  • Exposing myocardium-attached active absorbing nanoparticles of the present invention to MR can generate ROS in close proximity to the myocardium cells.
  • ROS molecules serve as signaling agents which stimulates various cells including myocardial cells.
  • Lavie et al [7b] exposed myocardial cells to 3.6 J/cm2 light flux and to hydrogen peroxide concentration of 12 ⁇ mol or 10.sup.15 molecules/cm.sup.3 . In both cases, they observed comparable and significant increase hydrogen peroxide and Ca+2 content, and stimulated cell functioning.
  • the present invention provides methods for infarcted myocardium rehabilitation preferably comprising the stages of: a) administering active absorbing nanoparticles to the myocardium tissue; (b) positioning an ultrasound source and a source of MR so as to couple their energy to the myocardium region; (c) exposing the myocardium region to simultaneous MR and ultrasound radiation so as to generate ROS therein; and (d) stimulating the myocardiocytes using the ROS as signaling molecules so as to improve the myocardial tissue functioning.
  • the microwave frequency is between 100 MHz and 3 GHz, and more preferably between 0.3 GHz and 1 GHz.
  • the electromagnetic source pulse width may optionally and preferably vary between 10 nanosecond and 30 milliseconds and more preferably between 0.01 and 10 microsecond.
  • the peak MR power density optionally and preferably ranges between 0.1 kW/cm2 to 1 MW/cm2, and more preferably between 1 kW/cm2 and 100 kW/cm2.
  • the nanoparticles are preferably administered through the blood vessels connected to the heart and more preferably attach using ligands to the damaged myocardial cells. In other aspects they are preferably released by maneuvering a catheter close to the myocardium, followed by their migration towards and preferable attachment to the myocardial cells. In yet other aspects, the catheter may optionally be equipped with suitable sensors to sense the region of malfunctioning myocardial tissue for optimized release of the nanoparticles.
  • the nanoparticles are preferably simultaneously exposed to MR and ultrasound radiation so as to generate nanobubbles near the nanoparticles and evolve the nanobubbles into microbubbles. Further exposing the myocardium would pulsate the microbubbles, thereby inducing microstreams that will migrate the ROS in the myocardium, thus minimizing localized ROS damage in the cells.
  • the ultrasound energy frequency for evolving the nucleation sites into microbubbles varies between about 300 kHz and 3 MHz and more preferably between 0.5 and about 2 MHz.
  • the average ultrasound energy levels may vary from about 0.5 Watt (W) per square centimeter (cm.sup.2) to about 20 W/cm.sup.2, with energy levels of from about 0.5 to about 2.5 W/cm.sup.2 being preferred.
  • the ultrasound energy is preferably coupled to the myocardium by placing one or more ultrasound transducers on the skin between the ribs in a region close to the myocardium.
  • the MR is preferably coupled to the myocardium by placing an end EM applicator on the skin, facing the myocardium.
  • the nanoparticles are preferably equipped with suitable ligands which attach to damaged myocardial cells.
  • the present invention (in some embodiments) preferably provides methods for preconditioning myocardial cells against events following MI event including but not limited to hardening mycardiocytes against oxidative stress, hardening vasculature against ischemia - perfusion damage, cytoprotective effect, thus alleviating post- reperfusion injury, following acute ischemia, including preserving the structure and orientation of contractile proteins in the cardiomyocytes, preservation of the structure and activity of mitochondria in cardiomyocytes,
  • Ischemic cardiac tissues may have their blood supply restored, using various treatments known in the art, such as coronary angioplasty.
  • the ischemic tissue may be rapidly reperfused with blood.
  • Such rapid reperfusion has been shown, at least in some cases, to result in post-reperfusion injury, i.e., damage induced by the reperfusion per se.
  • the injury may be caused, inter alia, by superoxides formed within the tissue due to a sharp increase in oxygen supply following the reperfusion procedure.
  • Several methods have been suggested to minimize ischemia — reperfusion injury. For example administering exogenous antioxidants such as seleno-organic compounds alleviate the toxic effect of the superoxides by scavenging the free radicals immediately upon formation.
  • Oron and Matkovich suggested exposing the ischemic myocardial tissue to optical radiation for cells preconditioning against I/R induced injury. They claim that the preconditioning may result from controlled generation of intracellular enzymes that can negate damaging ROS signaling, replenishing essential metabolites such as ATP, and controlled exposure of mitochondria to intracellular materials that would be generated following I/R event.
  • Lavie et al [7b] exposed cardiac cells to a dose of 3.6 J/cm2 light during 90.seconds.
  • the observed induced ROS effects were simulated by exposing the cells to 12 ⁇ mol of extracellular hydrogen peroxide. They observed 10% increase in Ca+2 intake. In other experiments using the same exposure, they observed cells proliferation.
  • ultrasound extracellular pulsating microbubbles may have similar effects to stimulating light dose which act mainly on intracellular components.
  • Myocardiocyte preconditioning is also not the only path for cell hardening against ischemia - reperfusion (I/R) injury.
  • Bertuglia [18a] exposed endothelial cells to periodic pulsed ultrasound in the presence of contrast agent microbubbles.
  • the present invention provides methods for preconditioning infarcted myocardium comprising the stages of: a) administering active absorbing nanoparticles to infarcted myocardium of a subject; (b) positioning an ultrasound source and a source of MR so as to couple their energy to the myocardium region; (c) exposing the myocardium to simultaneous MR and ultrasound radiation so as to generate nanobubbles near the nanoparticles, and then evolving the nanobubbles into microbubbles and pulsating them.
  • the infarcted myocardium tissue area is 10 cm.sup2 and its thickness is a few mm. Therefore the nanoparticles should be administered relatively uniformly into the myocardium volume.
  • the nanoparticles are administered to the infarcted myocardium region through blood vessel directly connected to the heart. Ultrasound may be applied before or during treatment to attain relatively uniform nanoparticles distribution within the infarcted myocardium volume.
  • the nanoparticles are released locally with a suitable catheter.
  • the catheter is equipped with a shaft carrying an array of needles in its distal end, where each needle can slide along its axis.
  • the needle array can be attached to a suitable means for axially sliding the needles including the mechanisms described by Nayak et al. in US Patent application 20060041243. Sliding the needles injects the nanoparticles throughout the infarcted myocardium volume.
  • the nanoparticles diffuse through the damaged myocardium tissue insterstitium using ultrasound enhanced diffusion.
  • the absorbing nanoparticles are preferably fabricated for preferential penetration into the myocardial cells. Exposing the nanoparticles to MR induces intracellular ROS generation which stimulates intracellular components such as the membrane and the mitochondria. In turn, the myocardium cells are preconditioned by processes equivalent to those observed following light induced preconditioning.
  • EXAMPLE 3 A suspension of nanoparticles of EXAMPLE 1 are administered to damaged myocardium tissue of human subject following MI event, by injection from a needle array, for accumulation in myocardial tissue to an averaged level of l*10.sup.7 nanoparticles /cm 3 .
  • the nanoparticles attach to the tissue cells as clusters, typically comprising 5-50 nanoparticles per cluster.
  • Two ultrasound transducers are placed onto the patient chest between the ribs.
  • An extracorporeal microwave antenna is placed facing the damaged myocardial tissue so as to couple adequate level of MR into the damaged myocardial tissue.
  • the damaged myocardial tissue with the attached nanoparticles is exposed simultaneously to a 2 GHz MR pulse train (50 pulses) and 1.5 MHz continuous ultrasound energy for 3 minutes .
  • Each MR pulse is 0.5 microsecond and peak intensity at the myocardial tissue 3 kW/cm2 while the ultrasound peak overpressure at the myocardium is 0.5 MPa.
  • the generated ROS break molecules from the inter and nanobubbles are generated near each nanoparticle.
  • parallel microbubbles evolved adjacent to a fraction of myocardial cells, according to the process described in EXAMPLE 1 above.
  • the myocardial cells are stimulated and respond by various ways including recovery into active myocardiocytes, converting scar tissue back into active myocardial tissue. After four consecutive sessions spaced two weeks apart, the treatment result is a significant decrease in infarcted myocardial tissue area, and enhanced functionality of the damaged myocardium leading to better heart functioning and reduced risk of repeated heart attack.
  • Section 4 Applications of absorbing nanoparticles for rehabilitating traumatized nerves.
  • This Section relates to treatments of traumatized nerves with the methods according to some embodiments of the present invention.
  • a first example relates to the use of ROS for stimulating neural cells towards regrowth, such as for example in the traumatized extremities of a subject.
  • a second example relates to treatments of traumatized nerves based on microbubbles interactions.
  • the present invention in some embodiments, preferably provides treatments for traumatized nerve rehabilitation.
  • Treatment includes but is not limited to: traumatized nerve regeneration, size reduction of scar tissue in traumatized nerve region, nerve cells enhanced differentiation or proliferation, nerve preconditioning- induced neuroprotection, improving functionality of traumatized nerves and system, and nerve cell biostimulation with one or more additional nerve rehabilitation clinical treatments.
  • Nerve disorders may be induced by external causes, diseases, and/or biological or physiological response related disorders.
  • External causes include, by way of example only, automotive related accidents, fall injuries, military and terror related injuries, internal surgery induced damage, sport activities, repetitive stressed nerve, etc.
  • Disease related nerve disorders include, by way of example only, neuralgia, radiculalgia, radiculopathy, sciatica, carpal and tarsal syndromes, compressive neuropathies, autonomic nervous system disorders, post surgical pain syndrome, causalgia, RSD, complex regional pain syndrome, post herpetic pain syndrome, chronic pain syndrome, diabetic neuropathy and peripheral neuropathy.
  • the blood-neural barrier is a barrier that maintains a precisely regulated microenvironment for reliable neuronal activities.
  • the BNB blocks all molecules except those that cross cell membranes by means of lipid solubility (including but not limited to oxygen, carbon dioxide, ethanol, and steroid hormones) and those that are allowed in by specific transport systems (such as sugars and some amino acids; see for example . Eriksson et. al., in US patent application 20070265203
  • the BNB comprises an extensive network of endothelial cells, pericytes, astrocytes and neurons that form functional "neurovascular units.”
  • the neurovascular unit is a conserved anatomical structure that is present at all sites where blood vessels meet neural tissues.
  • the neurovascular unit is composed of endothelial cells lining the inner surface of the vessel, perivascular pericytes that are tightly attached to the vessel, and an outer sheet of perivascular astrocytes. The presence of tight junctions between endothelial cells and specific BNB transporters ensures the BNB 's function as a selective diffusion barrier.
  • the present invention provides, in some embodiments, methods for nerve rehabilitation using absorbing nanoparticles which by exposure to MR generate ROS and microbubbles.
  • the methods provided by the present invention may optionally provide nerve rehabilitation through several main mechanisms including but not limited to: increasing the nerve cells functionality (e.g, action potential), cell proliferation (e.g., increased growth rate of basal lamina tube), nerve cell differentiation and reduction of scar tissue size around the traumatized nerves.
  • a cut generated along an axon due to trauma induces a cascade of events which normally lead to the destruction of the axon and its myelin envelope. The process involves secretion of excessive ROS and other signaling molecules.
  • a renervation i.e., growth on a new axon
  • a renervation process initiates. It starts with a growth of basal lamina tube with a growth cone.
  • a new myelin tube grows and in fact guides the lamina tube towards the target muscle or the next microglial cell (see for example Wang et al., in US patent application 20040186320).
  • ROS plays a major role in nerve rehabilitation. Recent evidence [2d] suggests that ROS might act as modulators (signaling agents) of neural processes, including synaptic transmission. The kinetics of ROS production may determine whether nerve cells will differentiate or proliferate [3d]. Therefore, controlled generation of ROS at traumatized nerve vicinity enhance its rehabilitation rate.
  • Nanoparticles generating ROS have a basic advantage over light based treatment since they can attach to nerves and axons using ligands or other attachment mechanisms. Thus, exciting these nanoparticles with suitable external energy will generate the desired amount of ROS near the target nerve cells without any adverse effects to other cells. As opposed to electrical stimulation methods, ROS based methods directly induce regeneration of neuronal conditions.
  • nanoparticles are advantageous for nerve rehabilitation since they can use ligand for preferential attachment to nerve cells or even traumatized nerve cells without affecting other cells.
  • certain types of nanoparticles can generate ROS following exposure to optical radiation.
  • most nerves are surrounded by thick tissue and thus the penetration of extracorporeal light, including NIR, into nerve sites is negligible. Attempts to treat such traumatized nerves using a catheter with light guiding capability or by a surgical procedure may be complex and hazardous.
  • the present invention provides methods for nerve rehabilitation by exposing absorbing nanoparticles to MR.
  • the absorbing nanoparticles are administered to traumatized nerves, preferentially attach to them and release ROS following exposure to MR.
  • the methods of the present invention are effective for nerve rehabilitation non-invasively within most locations within the body.
  • the nanoparticles may comprise suitable ligands for preferred attachment onto traumatized nerves.
  • Methods of introduction include the use of small (5 - 20 nm) nanoparticles coating the nanoparticles with lipids and other compositions which enable limited penetration through the BNB. However, these methods are characterized with slow throughput and are typically limited to specific BNB types. There is a need for a method that would enable significant nanoparticles and drug transport through the BNB using simple, non-invasive and safe procedures.
  • Fig. 2 describes one exemplary, illustrative but preferred embodiment of a method for enhanced transport of materials through the BNB (blood nerve barrier).
  • Stage I depicts a detailed cross section of a BNB region 203 near a traumatized nerve cell 255, comprising a microvessel 200 built from a plurality of curved endothelial cells 208 along its length, each connected by a tight junction 260, thereby forming a lumen 210 through which blood can flow.
  • the microvessel 200 may comprise one or more additional layer(s) 215 which further protect it from mechanical damage.
  • An microwave source 225 is preferably operable to expose the microvessel 200 to MR 230.
  • An ultrasound source 235 is optionally and preferably operable to expose the microvessel 200 to ultrasound energy 240.
  • FIG. 2B An enlarged view of the tight junction 260 region of microvessel 200 is illustrated in Fig. 2B.
  • one or more absorbing nanoparticle(s) 265 are preferably administered to the blood flowing through microvessel 200 whereas a significant fraction of them attach to the tight junction 260 within microvessel 200, more preferably using a suitable ligand and preferably as clusters.
  • Exposing the microvessel 200 to MR 230 and ultrasound energy 240 generates a nanobubble 250 around nanoparticle(s) 265 adjacent to the tight junction 260, through the process described above (stage II).
  • stage III The action of one or more expanding nanobubble(s) 250 adjacent to the tight junction 260 applies sufficient stress on the tight junction 260, and opens an initial gap 270 in it, which is sufficient for diffusion of additional nanoparticles 265 into the tight junction 260. (stage III).
  • the rehabilitation process of the traumatized nerve cell 255 is illustrated in Fig. 2C.
  • additional nanoparticle(s) 265 enter through the gap 270 extend it, and finally preferably temporarily open the gap 270 along its entire length.
  • nanoparticles 265 which diffused through the gap 270 attach to the target nerve cells and generate ROS 275 following exposure to the MR.
  • the ROS 275 flux stimulate the nerve cell 255, with enhanced metabolism, resulting in growth, sprouting 280 and eventually reconnecting to other nerve cells (stage III) and rebuilding the traumatized nerve network, resulting in functional rehabilitation of the organ comprising the traumatized nerve network.
  • the present invention provides methods for treating traumatized nerves using nanoparticle(s) 265 and bioactive material.
  • the nanoparticle(s) 265 are used for breaching the BNB by opening a gap 270 within the tight junction 260 enabling transport of bioactive composition suitable for rehabilitation of traumatized nerve cell(s) 255. Accordingly, the ultrasound energy 240 parameters and nanoparticle(s) 265 characteristics may be insufficient to open the gap 270 in the tight junction 260 for nanoparticle(s) 265 diffusion. However the gap 270 is sufficiently opened for transient transport of bioactive composition through the BNB to the traumatized nerve cell(s) 255.
  • the microwave frequency is between 100 MHz and 3 GHz, and more preferably between 0.3 GHz and 1 GHz.
  • the electromagnetic source pulse width may optionally and preferably vary between 10 nanosecond and 30 milliseconds and more preferably between 0.01 and 10 microsecond.
  • the peak MR power density optionally and preferably ranges between 0.1 kW/cm2 to 1 MW/cm2, and more preferably between 1 kW/cm2 and 100 kW/cm2.
  • the ultrasound energy frequency for evolving nucleation sites into nanobubbles varies between about 300 kHz and 3 MHz and more preferably between 0.5 and about 2 MHz.
  • the average ultrasound energy levels may vary from about 0.5 Watt (W) per square centimeter (cm.sup.2) to about 5 W/cm.sup.2, with energy levels of from about 0.5 to about 2.5 W/cm.sup.2 being preferred.
  • the present invention preferably provides methods for treating traumatized nerves using nanoparticle(s) 205 and bioactive material.
  • the nanoparticle(s) 205 are utilized for breaching the BNB by opening a gap 270 within the tight junction line 260 enabling transport of bioactive composition suitable for rehabilitation of traumatized nerve cell(s) 255.
  • the ultrasound energy 240 parameters and nanoparticle(s) 205 characteristics may be insufficient to open the gap 270 in the tight junction line 260 for nanoparticle(s) 265 diffusion.
  • the gap 270 is sufficiently opened for transient transport of bioactive composition through the BNB to the traumatized nerve cell(s) 255.
  • the present invention provides methods for traumatized nerve(s) rehabilitation comprising the stages of: a) administering absorbing nanoparticles to traumatized nerve cell(s); (b) positioning an MR soruce so as to couple its energy to the traumatized nerve vicinity; and (c) exposing the nerve vicinity to MR so as to generate ROS adjacent to the nerve cell(s).
  • the nanoparticles are simultaneously exposed to MR and ultrasound radiation so as to generate nanobubbles near the nanoparticles and evolve the nanobubbles into microbubbles.
  • the ultrasound energy frequency for evolving nucleation sites into microbubbles varies between about 300 kHz and 10 MHz and more preferably between 0.5 and about 5 MHz.
  • the average ultrasound energy levels may vary from about 0.5 Watt (W) per square centimeter (cm.sup.2) to about 20 W/cm.sup.2, with energy levels of from about 0.5 to about 2.5 W/cm.sup.2 being preferred.
  • the amount of active absorbing nanoparticles administered to the nerve vicinity, the MR operating parameters and ultrasound parameters are preferably optimized according to the type of nerve, the severity of trauma and the condition of surrounding tissue. In certain cases, the number of treatment sessions is increased without increasing microwave or ultrasound power in order to enhance the renervation while preventing ROS damage the cells at the treated nerve vicinity.
  • the present invention in some embodiments, preferably provides methods for treatment of neurological disorders that result from the overproduction of nitric oxide and other ROS types by neuronal and other cells.
  • overproduction may result from nerve damage following injury, ischemia and nerves disorders.
  • NMDA N- methyl-D-aspartate
  • NMDA receptors are also thought to be involved in long-term potentiation, central nervous system (CNS) plasticity, cognitive processes, memory acquisition, retention, and learning. Furthermore, the NMDA receptor is involved in a broad spectrum of CNS disorders. During brain ischemia caused by stroke or traumatic injury, excessive amounts of the excitatory amino acid glutamate are released from damaged or oxygen deprived neurons. This excess glutamate binds to the NMDA receptor which opens the ligand-gated ion channel thereby allowing Ca.sup.2+ influx producing a high level of intracellular Ca.sup.2+ .
  • Increased Ca2+ cellular uptake results in enhanced metabolism.
  • mitochondrial Ca2+ uptake upregulates energy metabolism, resulting in increased generation of ROS [5d].
  • moderately increasing the Ca2+ uptake results in cells including neural cell proliferation.
  • the Ca2+ uptake is increased dramatically, such as following a nerve trauma, it activates biochemical cascades resulting in extensive ROS production and in turn to protein, DNA, and membrane degradation, leading to cell death.
  • the extensive ROS generation is known as excitotoxicity, and responsible for many neurological events following nerve injury or ischemia.
  • Nerve Preconditioning is an acute procedure conducted following extensive tissue damage which involved also nerve trauma.
  • light induced nerve rehabilitation treatments are typicall conducted through multi-sessions and slow progressing treatment.
  • the laser treatment reduced the trauma induced inflammation and apparently blocked the NF-B signaling pathway, which believed to be responsible to the muscle trauma which follows nerve trauma.
  • ROS induced by light or by other mechanisms
  • ROS-dependent hypoxic preconditioning in cardiomyocytes appears to be related to superoxide transfer to the cytosol via mitochondrial anion channels.
  • ROS have been implicated in ischemic tolerance generated by "chemical" preconditioning of the brain. Superoxide radicals and H2O2 alone can 'precondition' neurons against glutamate toxicity or ischemia.
  • the present invention in certain embodiments, preferably provides methods for neuroprotecting nerve(s) following a traumatic event comprising the stages of: a) administering absorbing nanoparticles to traumatized nerve vicinity; (b) positioning an ultrasound source and a source of MR so as to couple their energy to the traumatized nerve vicinity; and (c) exposing the nerve vicinity to MR so as to generate ROS near the nerve cells, thus preconditioning them against damage induced by the traumatic event.
  • microbubbles are preferably generated by exposing the nanoparticles simultaneously to MR and ultrasound radiation. These microbubbles pulsate by the ultrasound radiation, induce microstreams and in turn translate the nanoparticles thus expose the nerve cells surfaces to a more uniform flux of ROS.
  • the pulsating microbubbles preferably act upon the target nerve cell(s), increasing the Ca2+ uptake rate and in turn preconditioning the target nerve cells against damage induced by the traumatic event.
  • the nanoparticles are simultaneously exposed to MR and ultrasound radiation so as to generate nanobubbles near the nanoparticles and evolve the nanobubbles into microbubbles. Further exposing the damaged nerve cell(s) to ultrasound would pulsate the microbubbles, thereby inducing microstreams that will migrate the nanoparticles in respect to the nerve cell(s), thus mimrnizing localized ROS damage in the cells.
  • Section 5 Applications of absorbing nanoparticles for moving fluids.
  • This Section relates to methods for moving small amount of fluids based on methods according to the present invention.
  • a first example relates to transport of fluids in a stent, using microbubbles generated by external energy source.
  • a second example relates to rapid mixing of fluids in small volumes using microbubbles generated by external energy source.
  • the main application of these processes is in the fields of implants and microfluidic devices of the type called lab-on-a-chip.
  • Implantable medical devices are often used for delivery of a bioactive composition, to an organ or tissue in the body at a controlled delivery rate over an extended period of time. These devices may deliver bioactive composition to a wide variety of bodily systems to provide a wide variety of treatments. The amounts of fluid being released in each release event are measured in a fraction of a nano-liter to micro-liter volumes.
  • Drug delivery from implant provides methods for externally controlled drug release from an implant.
  • the methods of the present invention enable delivery of bioactive compositions to a patient and selectively modulating, activating, or deactivating bioactive compositions after their delivery to targeted tissue regions adjacent the implanted medical device.
  • the typical amount of drug released from a single delivery unit may be measured by a fraction of nano-liter.
  • vascular stents One of the many implantable medical devices which have been used for local delivery of beneficial compositions is the vascular stent.
  • Vascular stents are typically introduced percutaneously, and transported transluminally until positioned at a desired location. These devices are then expanded inside the blood vessel, become encapsulated within the body tissue and remain a permanent implant.
  • Restenosis is a wound healing process that reduces the vessel lumen diameter by extracellular matrix deposition, neointimal hyperplasia, and vascular smooth muscle cell proliferation, and which may ultimately result in renarrowing or even reocclusion of the lumen.
  • the overall restenosis rate is still reported in the range of 25% to 50% within six to twelve months after an angioplasty procedure. To treat this condition, additional revascularization procedures are frequently required, thereby increasing trauma and risk to the patient.
  • Saul et al. in US Patent application 20050191708 teach a method for remotely moving fluids in a microfluidic device using MR induced heating.
  • a heat transforming material within the microfluidic device is heated by the MR and pushes the fluid through the cJhannel(s) to the desired destination.
  • the suggested scheme requires large MR dose beyond the level permitted to treat a human subject.
  • the present invention in some embodiments overcomes these drawbacks of the background art by providing methods for drug release from an implant, preferably using microbubbles generated and released from a reservoir comprising the bioactive composition.
  • the drug release unit comprises a reservoir filled with suitable bioactive composition and absorbing nanoparticles and a channel with an open or openable exit port. Exposing the release unit to MR and ultrasound generate one or more microbubbles within the reservoir. Each microbubble migrates through a channel towards the release opening and pushes a small amount of bioactive composition through the exit port.
  • the present invention provides methods for drug release from implant which comprise the stages of: (a) placing an implant comprising one or more drug release units and releasable bioactive composition within the desired location in the patient body, in which each release unit comprises absorbing nanoparticles in a reservoir filled with the bioactive composition and an open channel for release the bioactive composition; (b) exposing the implant to simultaneous MR and ultrasound energy sources thereby generating nucleation site(s) within the release unit(s); (c) evolving the nucleation site(s) into at least one microbubble through the action of the ultrasound radiation; and (d) releasing controlled amount of bioactive composition from the release unit(s) by the action of the migrating microbubble(s).
  • FIG. 3 shows an exemplary, illustrative but preferred embodiment of drug release methods from a drug carrying stent utilizing extracorporeal energy sources, according to the present invention.
  • Fig. 3A shows a detailed view of a diseased blood vessel 305 with a restenosis region 312.
  • a stent 300 comprising at least one micro-chamber 310 filled with bioactive composition is installed so as to improve blood flow within the blood vessel 305 adjacent to the stenotic region 312.
  • the stent 300 is optionally and preferably fabricated such that a free surface contour 370 is in a uniform close proximity to the blood vessel wall 308, after positioning.
  • An extracorporeal MR source 320 is preferably positioned and operable so as to expose stent 300 to MR 325 with suitable parameters.
  • a suitable extracorporeal ultrasound source 330 is also preferably positioned and operable to expose stent 300 to ultrasound energy 335 with suitable parameters.
  • Fig 3 B shows the drug release process from the micro-chamber 31 filled with bioactive composition 315.
  • At least one internal surface 340 of micro-chamber 310 comprises absorbing nanoparticles 345, preferably arranged as clusters, and fixated on the internal surface 340
  • a channel 350 preferably connects the micro-chamber 310 with a release port 355 on the stent contour surface 370. Exposure of absorbing nanoparticle(s) 345 to MR 325 and ultrasound energy
  • microbubble 360 preferably induces generation of one or more microbubble(s) 360 within the micro-chamber 310 through the process described above.
  • the microbubble(s) 360 grows through rectified diffusion under the ultrasound energy 335 until microbubble 360 detaches from the internal surface 340 and moves along the pressure gradient within the channel 350 to the release port 355. From release port 355, microbubble 360 is dissolved within the space 365 between the implant 300 and the blood vessel wall 308.
  • microbubble 360 pushes a finite volume of bioactive composition 315 for release through the release port 355 to the space between the stent 360 and the blood vessel wall 308, thus increasing the bioactive composition 315 concentration at close proximity to the stenotic region 312.
  • the increased concentration of bioactive composition 315 preferably reduces, at least temporarily, the proliferation of smooth muscle cells, which in turn reduces damage to the blood vessel wall 308.
  • the MR frequency is between 20 MHz and 3 GHz, and more preferably between 50 MHz and 1 GHz.
  • the MR source pulse width may optionally and preferably vary between 10 nanosecond and 30 milliseconds and more preferably between 0.01 and 10 microsecond.
  • the peak MR power density optionally and preferably ranges between 0.1 kW7cm2 to 1 MW/cm2, and more preferably between 1 kW/cm2 and lOO kW/cn ⁇ .
  • the ultrasound source frequency may be between about 20 kHz and 10 MHz and more preferably between 50 kHz and about 1 MHz.
  • energy levels may vary from about 0.5 Watt (W) per square centimeter (cm.sup.2) to about 20 W/cm.sup.2, with energy levels of from about 0.5 to about 2.5 W/cm.sup.2 being preferred.
  • the ultrasound energy may optionally and preferably comprise a single pulse, or a pulse train mode, or any other time sequence suitable for evolving a microbubble around absorbing nanoparticle(s) which are exposed simultaneously to appropriate light radiation pulse.
  • a drug release implant includes but not limited to: vascular grafts, stents, pacemaker leads, heart valves, implanted sensor, a drug implant for cancer treatment, and the like that are implanted in blood vessels, heart, tissue or non-tissue material, hi other aspects, the cells, tissue or non-tissue material to be treated by the released drug may also heated by coupling the ultrasound energy with the microbubbles released from the implant.
  • the heating and drag release timing may be adjusted by varying the MR and the ultrasound intensities and sequence.
  • the present invention provides in some embodiments methods for killing or capturing targeted entity flowing within the blood through the use of an implant.
  • targeted entity refers to but not limited to: diseased or malfunctioning cells, non-tissue material, organisms, viruses or foreign material particles.
  • the targeted entity comprises cancerous cells which are released to the blood and which are preferably removed before metastasis.
  • a device that will selectively kill the undesired targeted entity or capture them by using a suitable mechanism.
  • a method for detecting and killing or capturing target entities during their flow in a relatively large blood vessel comprising the following stages: (a) positioning one or more implants suitable for capturing or destruction of the cell or material in a suitable blood vessel; b) placing a suitable ultrasound source and an MR source close to the implant; c) administering specific markers entity (molecule, cluster or nanoparticle) suitable for attachment to the cell or material systemically to the subject body; d) detecting the marker entity with a suitable sensor positioned close to the implant; and e) operating the ultrasound and MR sources so as to non-invasively activate the implant and kill or capture the cell or molecule.
  • specific markers entity molecule, cluster or nanoparticle
  • one or more microbubble(s) is released from the implant and attach to the marker entity with its attached target entity.
  • the released microbubble may carry suitable material which promotes its attachment to the marker entity.
  • a microbubble diversion device such as directional ultrasound transducer, diverts the microbubble(s) from the main flow towards an implanted dump chamber or sacrificed blood vessel.
  • the released microbubble(s) attach to the marker entity and are disrupted by a suitable ultrasound pulse or shock wave, and in turn disrupt and kill the attached target entity.
  • the present invention in some embodiments provides methods for rapid mixing in microfluidic devices using microbubbles generated near absorbing nanoparticles.
  • Microfluidic devices especially lab-on-a-cbip devices, find increasing use in various applications ranging from drug discovery to molecular diagnostics.
  • the accuracy of the measured parameters strongly depends on proper mixing of the reagent and the sample liquids.
  • the throughput of such lab-on-a-chip devices employed in mass processes such as drug discovery depends strongly on the mixing rate.
  • the mixing rate of two fluids flowing within a micro-channel may take many seconds and consume relatively large volumes of sample and reagent.
  • Mixing may be conducted by a micropump installed within the lab-on-a-chip device.
  • active devices significantly increase the device cost.
  • passive mixing based on a labyrinth installed on the lab-on-a-chip to enhance the mixing. These methods induce significant pressure drop and affect the overall device cost.
  • Zwaan et al, [Ie] used alaser induced microbubble to mix two different fluids flowing through a small microfluidic channel]. They exposed two liquids flowing in parallel within a 0.1 mm wide channel to a pulse of focused laser beam, inducing microbubble within the channel. They demonstrated complete mixing of the liquids within 20 milli seconds.
  • this method requires a high quality objective for each mixing site within the lab-on-a-chip being processed.
  • a typical drug discovery lab-on-a-chip comprises multiple mixing sites, and would lead to a high cost and complicated processing assembly for such devices.
  • the present invention in some embodiments provides methods for improved process for mixing small volumes of liquid on demand in a micro chamber, comprises the stages of: (a) bringing the liquid(s) to be mixing chamber comprising at least one surface with fixated absorbing nanoparticles; (b) exposing the surface to simultaneous MR and ultrasound energy thereby generating at least one nucleation site on the surface; (c) evolving the nucleation site into at least one microbubble by the action of the ultrasound radiation; and e) further exposing the surface to ultrasound radiation and expanding the microbubble until it touches at least one more surface of the mixing chamber.
  • the fluid movement resulting from the microbubble expansion and bouncing induce rapid rotational flow, resulting in rapid mixing.
  • the advantage of the mixing methods according to the present invention is by reducing the cost and complexity of the processing head assembly.
  • the nanoparticles of the present invention can generate a microbubble using incident laser energy density below 10 mJ/cm.sup.2..
  • an integrated microlens on the transparent cover is sufficient for inducing a microbubble.
  • one or more micro-chambers are integrated into a lab-on-a-chip with a transparent cover with integrated microlenses.
  • the processing head assembly may preferably comprise a single laser with suitable optics for dividing the laser beam into multiple beamlets, each incident on one of the microlens(es).
  • the fixated nanoparticles induce nucleation sites which evolve to a microbubble by the ultrasound energy.
  • the microbubble expands, rebounds from the chamber wall, and in turn mixes the liquids within the chamber as described above. Section 6 - Applications of absorbing; nanoparticles for water treatment.
  • This Section relates to water treatments using the methods according to the present invention. For example absorbing nanoparticles are used to generate microbubbles which in turn serve as electric discharge centers which in turn release ROS which decontaminate water from contaminating materials.
  • the present invention in some embodiments preferably provides methods for water decontamination including, production of pharmaceuticals, decontaminating paper industry effluent discharge, removal of odor and contaminant from industrial discharging liquid and treatment of other liquid effluent.
  • the contaminating materials may include: dissolved organic contaminants, sanitary products, industrial and agricultural residues, bacteria, viruses, fungus, odor materials and mixtures thereof.
  • the decontaminants may be neutralized or chemically modified by processes which break down or modify the undesired molecules. Previous decontamination processes include exposure to UV radiation, ozone generation and bubbling, exposure to intense ultrasound, electrical glow discharges and combination thereof.
  • effective chemical decontamination requires intimate contact of the short lived active compounds (e.g., hydrogen peroxide and radicals) with the liquid volume. This can be done for example by generating small bubbles comprising active molecules such as ozone.
  • the present invention overcomes the above drawbacks of the background art by providing methods in some embodiments for microbubble generation near absorbing nanoparticles exposed to MR and ultrasound.
  • the methods have many advantages over previous methods, including but not limited to the larger density of microbubbles (e.g., 3*10.sup.5 per cubic cm), compared to densities attainable by other methods; microbubbles have very high specific surface area (e.g, 30 square meter per cubic meter); consumed energy is much lower compared to other methods and may be significantly less than 1 kWh/per cubic meter.
  • the present invention in some embodiments provides methods for generating a non thermal plasma in the form of discharge in microbubbles within a contaminated liquid.
  • the liquid is introduced in to a chamber having at least one electrode, connected to an electric current source or MR source.
  • Microbubbles are generated by the methods of the present invention, hi turn, the voltage or MR power is increased until a glow discharge is ignited within at least a portion of the microbubbles.
  • Electrons are generated in the non-thermal plasma within the bubbles causing ionization and dissociation of gaseous and/or vapor molecules within the microbubbles.
  • highly reactive species such as ROS are generated within each microbubble and diffuse to its water interface.
  • methods for treatment of aqueous liquids comprising undesired materials based on discharge generated within microbubbles, comprising the stages of: (a) contacting the aqueous mixture comprising material(s) to be removed, neutralized or modified with one or more surfaces coated with absorbing nanoparticles layer; (b) exposing the reactive surfaces to simultaneous MR and ultrasound radiation thereby generating dense microbubbles cloud within the liquid volume; and (c) generating discharge within the microbubbles by means of MR and/or a separate electric current source.
  • the interactions of the discharge product at the microbubble interface with the material(s) to be modified in the liquid environment causes removal, neutralization and/or modification of the material(s).
  • the object(s) are positioned in a reaction enclosure and comprise at least one surface coated with absorbing nanoparticles.
  • the flow pattern within the interaction volume sweeps the microbubbles generated on the surface(s) so as to uniformly fill the reaction enclosure.
  • at least a portion of the absorbing nanoparticles are fixated on relatively large non-conductive flakes positioned within the interaction region. In other embodiment the flakes may flow with the liquid, screened and recycled into the interaction region.
  • a conductivity enhancing agent is added to the aqueous medium to enhance its electrical conductivity such as alkaline or acidic material, organic salts or inorganic salts, e.g. NaCl, NaOH, CaCO3, HCl 5 . etc.
  • a surfactant composition may be added to the aqueous medium for lowering the surface tension of the liquid and help stabilizing the microbubbles during evolution.
  • suitable materials are added to the contaminated liquid for enhancing the process rate, such as oxygen, ozone, catalysts and additives for buffering the pH.
  • the MR frequency is between 20 MHz and 3 GHz, and more preferably between 50 MHz and 1 GHz.
  • the MR source pulse width may optionally and preferably vary between 10 nanosecond and 30 milliseconds and more preferably between 0.01 and 10 microsecond.
  • the peak MR power density optionally and preferably ranges between 0.1 kW/cm2 to 1 MW/cm2, and more preferably between 1 kW/cm2 and 100 kW/cm2.
  • the ultrasound source frequency may be between about 20 kHz and 10 MHz and more preferably between 50 kHz and about 1 MHz.
  • energy levels may vary from about 0.5 Watt (W) per square centimeter (cm.sup.2) to about 20 W/cm.sup.2, with energy levels of from about 0.5 to about 2.5 W/cm.sup.2 being preferred.
  • the ultrasound energy may optionally and preferably comprise a single pulse, or a pulse train mode, or any other time sequence suitable for evolving microbubbles around absorbing nanoparticles which are exposed simultaneously to appropriate MR.
  • the initiation of a glow discharge and generation of plasma within the microbubbles may be assisted by a pulsed electric current supply, or shock wave source coupled with the region of microbubbles generation. These methods may reduce the threshold electric current or MR intensity required to ignite a glow discharge in at least a portion of the microbubbles.
  • the microbubbles may be generated throughout the interaction region or within a sheath close to an electrode couples the discharge electric current or the MR to the interaction region.
  • the reaction chamber comprises at least one conductive object connected to the electric current or MR source.
  • the surface may be an envelope conductor, a center rod electrode or any other configuration suitable for efficient coupling of electric energy or MR with the microbubbles.
  • the EMF within the interaction chamber may vary between 1 - 100 kV/m.
  • the localized pulsed electric field for initiating the glow discharge may be 0.1 - 10 MV/meter.
  • the electrical pulse timing may be synchronized with the microbubble concentration threshold which may be monitored by ultrasound reflections received by an ultrasound transducer, or with a shock wave generated by a suitable source.
  • Oxygen-Glucose Deprivation Tolerance Induction Through Abbreviated Neurotoxic Signaling," Am. J. Physiol. Cell Physiol. V n6 (2003).

Abstract

Des nanoparticules absorbant le rayonnement électromagnétique peuvent être mises en œuvre pour la génération d'espèces excitées lors d'une exposition à un rayonnement électromagnétique dans la gamme des micro-ondes. Selon d'autres formes de réalisation de la présente invention, on propose des procédés de génération de microbulles proches des nanoparticules par interaction de la nucléation induite par les espèces excitées avec les ultrasons. Selon d'autres formes de réalisation encore de la présente invention, on propose des procédés et appareils, qui utilisent de telles nanoparticules pour des traitements basés sur la génération contrôlée d'espèces excitées.
PCT/IL2007/001462 2006-11-27 2007-11-27 Génération de ros par les nanoparticules, microbulles et leur utilisation WO2008065652A2 (fr)

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ITVR20130037A1 (it) * 2013-02-13 2014-08-14 Giglio Antonio Del Trattamento di tessuti biologici mediante onde d'urto ed impulsi di radiofrequenza contrapposti.
WO2019032771A1 (fr) * 2017-08-09 2019-02-14 University Of Cincinnati Nanotransporteurs à base d'acide undécylénique pour l'administration ciblée de médicament
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Cited By (15)

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EP2332612A4 (fr) * 2008-09-30 2012-03-07 Panasonic Elec Works Co Ltd Procédé de réduction du degré d'activation de cellules, et appareil pour ledit procédé
EP2332612A1 (fr) * 2008-09-30 2011-06-15 Panasonic Electric Works Co., Ltd. Procédé de réduction du degré d'activation de cellules, et appareil pour ledit procédé
US8192429B2 (en) 2010-06-29 2012-06-05 Theravant, Inc. Abnormality eradication through resonance
CN103764226A (zh) * 2011-08-12 2014-04-30 保罗·科西斯梅迪亚 用于治疗包括病毒和细菌的病原体的方法和设备
EP2741822A4 (fr) * 2011-08-12 2015-05-06 Paul Csizmadia Procédé et appareil pour le traitement de pathogènes dont les virus et bactéries
ITVR20130037A1 (it) * 2013-02-13 2014-08-14 Giglio Antonio Del Trattamento di tessuti biologici mediante onde d'urto ed impulsi di radiofrequenza contrapposti.
US20200000917A1 (en) * 2017-02-22 2020-01-02 The General Hospital Corporation Methods of photochemical treatment for wound healing
WO2019032771A1 (fr) * 2017-08-09 2019-02-14 University Of Cincinnati Nanotransporteurs à base d'acide undécylénique pour l'administration ciblée de médicament
US11717578B2 (en) 2017-08-09 2023-08-08 University Of Cincinnati Undecylenic acid-based nanocarriers for targeted drug delivery
CN109701014A (zh) * 2019-01-18 2019-05-03 中国科学院理化技术研究所 一种用于微波动力治疗肿瘤的微球及其制备方法和应用
CN109701014B (zh) * 2019-01-18 2021-07-27 中国科学院理化技术研究所 一种用于微波动力治疗肿瘤的微球及其制备方法和应用
US20200254273A1 (en) * 2019-02-07 2020-08-13 Weinberg Medical Physics, Inc. System, methodologies and components for skin sculpting with magnetic particles
US11701522B2 (en) * 2019-02-07 2023-07-18 Weinberg Medical Physics Inc System, methodologies and components for skin sculpting with magnetic particles
DE102020129157A1 (de) 2020-11-05 2022-05-05 Lavenir Bioscience Ag Vorrichtung zur Entfernung wenigstens eines Objekts oder einer Agglomeration von Objek-ten aus der Haut
WO2024076696A1 (fr) * 2022-10-05 2024-04-11 General Electric Company Accélération de la cicatrisation cutanée au moyen d'ultrasons focalisés non invasifs

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