Patent Application
20140219922
Not Applicable
Not Applicable
Not Applicable
1. Field of Invention
The present invention relates to the methods of non-invasive control of the activation of light-sensitive bioactive molecules inside live organisms for the purpose of research or therapy.
2. Discussion of Prior Art
Recent advancements in the development of light-sensitive bioactive molecules and biosensors capable of changing their functional state in response to light irradiation as well as rapid progress in optogenetics provided the researchers invaluable tools for studying numerous biological, physiological and pathophysiological processes and for influencing these processes in the in vitro experimental settings (i.e., in various types of cells and tissues out of organisms) and even in experimental animals in vivo. The light-sensitive bioactive compounds can be artificial or natural.
The first class of artificially synthesized light-sensitive compounds is represented by composite molecules consisting of fluorescence group coupled with active group of which the former serves as a light sensor whereas the latter can bind various exogenous or endogenous bioactive molecules. Upon irradiation with the light of certain wavelength fluorescence group absorbs the quantum of that light and in response emits quantum of light with longer wavelength (i.e., less energetic). The spectrum of light absorption and/or emission can change if active group binds or interacts with other molecules. Such light-sensitive compounds are usually used as reporters of the concentration of certain bioactive molecules or ions within the cells or out of the cells. The best know examples are various types of fluorescent Ca2+-sensitive dyes capable of reporting concentration of this physiologically important cation in various cellular compartments.
The second class of synthetic light-sensitive compounds, known as caged compounds, have a photolabile (i.e., subject to photochemical or photophysical reactions) protecting group attached to a significant functional group so as to render the whole molecule biologically inert. Light irradiation removes the protecting group to release the biologically active group. Photorelease of biologically active molecules potentially affords enhanced control over their administration and activation or inhibition of their biological target(s).
Fluorescence chemical groups can be also attached to specific antibodies enabling labeling of various cellular proteins and their visualization.
Finally, identification and cloning of numerous bioluminescent and light-sensitive proteins from various organisms such as, aequorin from luminescent jellyfish, luciferases from bacteria and fireflies, green fluorescent protein (GFP) from jellyfish or sea pansy, channelrhodopsins from green algae, halorhodopsin from halobacteria, etc. gave rise to the whole new avenue of optogenetics (Deisseroth (2011) Nat. Methods 8, 26-29). By inserting recombinant genes for these proteins alone or in combinations with other genes optogenetics allows visualization of various structures and processes as well as controlling cells' behavior in desired direction. For instance, the blue-light sensitive plasma membrane ion channel-forming protein, channelrhodopsin-2 (ChR2, activation wavelength 470 nm), and the yellow light-activated chloride pump halorhodopsin (peak absorbance at 570 nM) expressed together enable multiple-color optical activation and silencing of neural activity with high precision (Rogan & Roth (2011) Pharmacol. Rev. 63, 291-315, Peron & Svoboda (2011) Nat. Methods 8, 30-34). Genetic engineering yielded multiple isoforms of light-sensitive proteins with different optical and functional properties (e.g., Hegemann & Möglich (2011) Nat. Methods 8, 39-42).
Although biomedical research utilizing light-sensitive synthetic compounds and optogenetic approaches is now widespread in the in vitro settings and in vivo animal experimentation, enormous opportunities that such tools provide for diagnostic and therapeutic purposes in humans are largely hampered by technical difficulties for non-invasive delivering of the necessary light stimuli to the required sites within the human body. Because of optical non-transparency of the tissues of higher vertebrates, currently light is delivered in experimental animals only using invasive procedures consisting in implanting portable light source (U.S. Pat. No. 5,445,608, August 1995) or fiber optic light guides which one end is positioned within the targeted area inside the body and another end is sticking out of the body. Besides, permanent implantation of the light-guide reduces versatility of the light-mediated manipulations by only one site to which guide is targeted without the possibility of effectively transferring illumination to other sites on demand dictated by diagnostic or therapeutic purposes. Thus, a novel nanotechnology-based strategy enabling non-invasive delivery of light to any site within the body is required for effective implementation of optical methods in clinics.
Recent progress in the development of nanoscale scintillators (phosphors) which absorb X-rays and emit visible light (U.S. Pat. No. 6,576,156, June 2003; U.S. Pat. No. 8,137,588, March 2012; Nikl (2006) Meas. Sci. Technol. 17, R37-R54; Liu et al (2008) Appl. Phys. Lett. 92, 043901; Morgan et al (2009) Radiat. Res. 171, 236-244) provides a novel approach to overcome the problems of reaching deep tissues by exploiting the high penetrating potential of ionizing X-radiation combined with local emission of visible light. So far this approach is most considered for photodynamic therapy (PDT) of cancer and cancer imaging (U.S. Pat. No. 8,328,785, December 2012; U.S. Pat. No. 7,538,329, May 2009; U.S. Pat. No. 7,018,395, March 2006; Chen & Zhang (2006) J. Nanosci. Nanotechnol. 4, 1159-1166; Liu et al (2008) Appl. Phys. Lett. 92, 043901; Morgan et al (2009) Radiat. Res. 171, 236-244). PDT is based on use of photosensitizers which need to be delivered to pathological tissue and which interaction with light results in the generation of cytotoxic species, such as singlet oxygen (1O2), free radicals and peroxides, that attack key structural entities within the targeted cells. Using scintillator nanoparticles not only provides the means of photosensitizers activation deep in the tissues, but also enables combination of radiotherapy with photodynamic therapy leading to significant enhancements in therapeutic cytotoxic effect on cancer cells (Morgan et al (2009) Radiat. Res. 171, 236-24). Using another type of penetrable electromagnetic irradiation, radio waves, also enabled remote heating of iron oxide nanoparticles targeted to the endogenous heat-sensitive TRPV1 channel via TRPV1-specific antibodies and stimulation of TRPV1-dependent insulin release from the tumors and lowering of blood glucose in mice (Stanley et al (2012) Science 336, 604-608), indicating that penetrable electromagnetic radiation represents promising strategy for reaching deep tissue layers.
A method for delivering visible light stimuli to any part inside the body of higher animals including humans with the purpose of activating light-sensitive bioactive compounds or photosensitive recombinant proteins is based on systemic infusion or local injection of scintillator-based nanoparticles. Nanoparticle core is made from scintillator material used to visualize X-ray beams with the protective coating which ensures aqueous solubility, non-toxicity, biocompatibility and prevention of aggregation and non-specific binding. Linkage to nanoparticles of targeting agents such as antibodies, aptamers will further enable directed accumulation of nanoparticles at the required site(s) within the body. Focused irradiation of the site with the highly penetrable X-rays will cause nanoparticles to emit visible light which will activate light-sensitive bioactive molecule(s) within this site causing sought therapeutic effect.
Various other objects, features and attendant advantages of the present invention will be more fully appreciated and the same becomes better understood from the following detailed description when considered in connection with the accompanying drawings in which like reference characters designate like or corresponding parts wherein:
The object of the invention is to devise method for non-invasive activation of light-sensitive bioactive molecules or recombinant proteins inside live organisms for the purpose of research or therapy. The proposed solution presents unsurpassed advantages over prior arts in that: 1) it is non-invasive; 2) it can be targeted to any specific site within animal or human body; 3) it can be repeatedly applied.
This object is attained by creating a nanoparticle which core is made of scintillator material that absorbs highly penetrable X-rays and in response emits visible light. The nanoparticle core is covered with biocompatible protective coating, and on its surface may bear conjugated antibodies enabling targeting to specific proteins. The type of antibody is selected such as to recognize either native surface membrane proteins of targeted cells or recombinant membrane proteins heterologously expressed in targeted cells using in vivo gene transfer technologies. With antibodies against native surface membrane proteins the scintillator nanoparticles can be used to uncage bioactive compounds such as, neurotransmitters, nucleotides, bioactive amines, calcium, pharmacological therapeutic agents, etc. in the vicinity of targeted cells or to activate foreign light-sensitive protein heterologously expressed in target cells. With antibodies against foreign light-sensitive plasma membrane proteins, such as ChR2 and/or halorhodopsin nanoparticles can be used to activate these proteins by direct resonance energy transfer as well as to uncage bioactive compounds in the vicinity of targeted cells. Since ChR2 represents plasma membrane light-activated Ca2+-permeable, cationic channel (Nagel et al (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 13940-13945), whereas halorhodopsin is a light-driven ion pump, specific for chloride ions (Lanyi (1990) Physiol. Rev. 70, 319-330), use of scintillator-based nanoparticles will enable remote, X-ray-mediated control of transmembrane ion fluxes in the cells these proteins are heterologously expressed in. Altering transmembrane ion fluxes will in turn affect excitability of electrically excitable cells (neurons, various types of muscle cells) cell or intracellular ionic homeostasis of non-excitable cells (glial, epithelial, stromal), which can be exploited for scientific or therapeutic purposes.
Simplified diagram of scintillator-based nanoparticle is presented in
Operations of X-ray, scintillator-based nanoparticle and light-sensitive bioactive molecules in the vicinity of target cell are presented in
Scintillator-based nanoparticles 10 are delivered to target cells 20 by either direct injection into required site of the body or via infusion to the bloodstream. Depending on their pharmacokinetics, bioactive caged compounds 30 is administered by the same or different rout before, together or after nanoparticles to ensure simultaneous presence of both nanoparticles and caged compounds in the vicinity of target cells. If photosensitive proteins are used along or instead of caged compounds as therapeutic or research tools, before administering of nanoparticles in vivo gene transfer technologies (Kay (2011) Nat. Rev. Genet. 12, 316-328) are employed to selectively express in target cells foreign photosensitive plasma membrane 21 proteins 22. Antibodies 13 conjugated to nanoparticle permit nanoparticle docking to the native surface proteins 23 of the cell or to the heterologously expressed foreign photosensitive protein 22 thereby increasing targeting specificity. X-rays 41 from X-ray source 42 are focused on target site within the body. Nanoparticle 10 absorbs X-ray and in response emits visible light 43 with the wavelengths, which must coincide with the wavelength for uncaging of caged compound 30 to cause dissociation of its bioactive constituent 31 from photolabile protecting group 32 or to activate photosensitive proteins 22. Free bioactive constituent of caged compound 31 or activated foreign photosensitive protein 22 in turn produce desired biological effect on target cells.
Currently a variety of scintillator materials with emission wavelength ranging from violet (˜400 nm) to red (˜700 nm) light are available (Nikl (2006) Meas. Sci. Technol. 17, R37-R54; Morgan et al (2009) Radiat. Res. 171, 236-244) enabling covering of absorption spectra of any caged compound or photosensitive protein.
No alternative embodiments are proposed.
Thus the reader will see that the proposed invention for using scintillate-based nanoparticles presents the most effective way for in vivo control of light-sensitive bioactive molecules ensuring specific non-invasive targeting of the cells within deep tissue layers of live organisms with the purpose of research or therapy.
The unsurpassed advantages and universality of the proposed method can lead to the development of new therapies and correctional treatments targeting any population of cells, organ or system within human body via localized, X-ray-stimulated released on demand of caged pharmacological agents and other bioactive compounds or activation of photosensitive recombinant proteins.
While the above description contains a number of specifics, these should not be construed as limitations on the scope of the invention, but rather as an exemplification of possible embodiment thereof. This especially relates to the design of composite scintillator-based nanoparticle and type of recombinant photosensitive protein being the object for X-ray-mediated light-induced activation. If the proposed method is to be used for simultaneous X-ray-mediated release of two or more caged compounds and/or activation of photosensitive proteins, co-administering of several scintillator-based nanoparticle with different wavelengths of emitted light should be employed. Alternatively, co-administering of additional fluorescence nanoparticle(s) along with single scintillator-based nanoparticle will be required. In this case fluorescence nanoparticle must be excited by the light wavelength emitted by scintillator-based nanoparticle and in turn emit the light with lower wavelength (i.e., less energetic) which will uncage the second caged compound or activate the second recombinant photosensitive protein. Increasing the number of caged compounds and/or photosensitive proteins to be simultaneously activated by X-ray irradiation will require administering of several scintillator-based nanoparticles or co-administering of several fluorescence nanoparticles with single scintillator-based nanoparticle. The co-administered fluorescence nanoparticles will serve the purpose of reemitting light of progressively longer wavelengths to activate multiple light-sensitive bioactive molecules.
Accordingly, the scope of the invention should be determined not by the embodiment(s) illustrated, but by the appended claims and their legal equivalents.
