WO2015106173A1

WO2015106173A1 - Compositions and methods for heating nano particles

Info

Publication number: WO2015106173A1
Application number: PCT/US2015/010922
Authority: WO
Inventors: Wasim H. CHOWDHURY; Anilchandra ATTALURI; Ronald Rodriguez; Robert Ivkov
Original assignee: The Johns Hopkins University
Priority date: 2014-01-10
Filing date: 2015-01-09
Publication date: 2015-07-16

Abstract

The present disclosure provides compositions and methods for the hyperthermal activation of iron nanoparticles both in vitro and in vivo. According to one or more embodiments, light in the near infrared range (e.g., 650nm and above) may be used to heat iron nanoparticles in vivo and in vitro. For example, LumiNIR, which is a high energy light source developed for the purpose of medical and animal imaging, may be used to efficiently heat iron nanoparticles in vivo and in vitro, and hyperthermal activation of such nanoparticles may be used to sensitize various tissues for therapeutic or surgical treatment.

Description

COMPOSITIONS AND METHODS FOR HEATING NANO PARTICLES

RELATED APPLICATION

The present application claims priority to, and the benefit under 35 U.S.C. § 119(e) of U.S. provisional patent application No. 61/925,960, entitled "COMPOSITIONS AND METHODS FOR HEATING NANOPARTICLES" filed on January 10, 2014. The entire contents of the aforementioned patent application are incorporated herein by this reference.

FIELD OF THE INVENTION

The present disclosure relates to compositions and methods for heating nanoparticles in vitro and in vivo. More particularly, the present disclosure relates to thermal heating of iron nanoparticles.

BACKGROUND OF THE INVENTION

Superparamagnetic iron oxide nanoparticles have been widely used in biomedical research because they possess useful magnetic properties, are relatively non-toxic, and possess a high degree of biocompatibility. Such nanoparticles have been used for drug delivery, medical imaging, cell targeting, and hyperthermia therapy. They ability to use iron oxide nanoparticles for the hyperthermal treatment of certain types of cells (e.g., cancer cells) is of particular interest since the side effects associated with such treatment are significantly lower than those associated with more radical treatments such as, for example, chemotherapy or radiotherapy. Typically, magnetic hyperthermia of iron oxide nanoparticles is caused by dipole relaxation induced by an alternating magnetic field (AMF). Once heated, locally injected iron oxide nanoparticles can generate sufficient heat in a specific region of an animal to specifically destroy tumors. Advantageously, such hyperthermal treatment poses relatively little danger (e.g., cellular damage) to surrounding healthy tissues. The local temperature of the injected iron oxide nanoparticles can also be controlled by the power of the AMF.

Unfortunately, the use of an AMF to heat iron oxide nanoparticles requires high current and voltage due to large air volume within the applied field. Moreover, such AMFs cannot easily be focused, making the use of this technique for heating iron oxide nanoparticles in vivo very difficult. Accordingly, there is an urgent need for compositions and methods of efficiently targeting iron oxide nanoparticles in vitro and in vivo for hyperthermal activation. SUMMARY OF THE INVENTION

By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

By "ameliorate" is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

By "alteration" is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.

By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide.

Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean "includes,"

"including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. "Detect" refers to identifying the presence, absence or amount of the analyte to be detected.

By "detectable label" is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.

By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include [insert]

By "effective amount" is meant the amount required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject.

Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

The invention provides a number of targets that are useful for the development of highly specific drugs to treat a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

"Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

By "inhibitory nucleic acid" is meant a double-stranded RNA, siRNA, shR A, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. For example, an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids delineated herein.

By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

By "marker" is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.

As used herein, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing, or otherwise acquiring the agent.

"Primer set" means a set of oligonucleotides that may be used, for example, for PCR. A primer set would consist of at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 80, 100, 200, 250, 300, 400, 500, 600, or more primers.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, "nested sub-ranges" that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

By "reduces" is meant a negative alteration of at least 10%, 25%, 50%, 75%, or

100%.

By "reference" is meant a standard or control condition.

A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.

By "specifically binds" is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.

Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30°C, more preferably of at least about 37°C, and most preferably of at least about 42°C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25°C, more preferably of at least about 42°C, and even more preferably of at least about 68°C. In a preferred embodiment, wash steps will occur at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68°C in 15 mM aCl, 1.5 mM trisodium citrate, and 0.1% SDS.

Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et ah, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine;

aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e^"3 and e^"100 indicating a closely related sequence.

By "subject" is meant a mammal, including, but not limited to, a human or non- human mammal, such as a bovine, equine, canine, ovine, or feline.

As used herein, the terms "treat," "treated," "treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated. Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a," "an," and "the" are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the nature and desired objects of the present invention, reference is made to the following detailed description taken in conjunction with the accompanying drawing figures wherein like reference characters denote corresponding parts throughout the several views and wherein:

FIGS. 1A-1C depict various views of a light source according to an illustrative embodiment of the invention.

FIGS. 2A and 2B depict medical devices according to illustrative embodiments of the invention.

FIG. 3 depicts a method of illuminating an object of interest according to an illustrative embodiment of the invention.

FIG. 4 depicts a prototype light source according to an illustrative embodiment of the invention.

FIG. 5 depicts a graph showing thermal heating of nanoparticles over time in vitro according to an illustrative embodiment of the invention.

FIG. 6 depicts a graph showing thermal heating of nanoparticles over time in vivo according to an illustrative embodiment of the invention. DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides compositions and methods for the hyperthermal activation of iron nanoparticles both in vitro and in vivo. According to one or more embodiments, light in the near infrared range (e.g., 650nm and above) may be used to heat iron nanoparticles in vivo and in vitro. For example, LumiNIR, which is a high energy light source developed for the purpose of medical and animal imaging, may be used to efficiently heat iron nanoparticles in vivo and in vitro, and hyperthermal activation of such nanoparticles may be used to sensitize various tissues for therapeutic or surgical treatment. For example, it is contemplated within the scope of the invention that the iron oxide nanoparticles may be targeted to specific tissues such as, for example, a tumor, and then subject to hyperthermal activation in order to destroy the tumor.

The present invention provides methods of treating disease and/or disorders or symptoms thereof which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising nanoparticles described herein to a subject (e.g., a mammal such as a human). Thus, one embodiment is a method of treating a subject suffering from or susceptible to a disease or disorder or symptom thereof such as cancer. The method includes the step of administering to the mammal a therapeutic amount of a compound herein sufficient to treat the disease or disorder or symptom thereof by hyperthermal activation of the compound, under conditions such that the disease or disorder is treated.

The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a compound described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).

As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

The therapeutic methods of the invention in general comprise administration of a therapeutically effective amount of the compounds herein, such as a compound of the formulae herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like).

Light Sources for Heating Nano Particles

Referring now to FIG. 1A, a light source 100 is provided and may be used to heat nanoparticles as described herein. The light source 100 includes a housing 102 including at least one wall 104 defining an aperture 106. The aperture 106 is adapted and configured to receive an optical fiber 108 along an axis 110. One of ordinary skill in the art will readily appreciate that certain portions of housing 102 are omitted from FIG. 1 so that the internal structure of the light source 100 can be viewed. The light source 100 also includes one or more lasers 1 12 positioned within the housing such that each of the lasers 1 12 is positioned at an angle with respect to the axis 110 and may generate a beam 1 14 focused on a common focal point 116 along the axis 110, within the housing 102, and offset from the aperture 106.

Light source 100 advantageously allows transmission of light to the outside world only through the optical fiber 108 and precludes accidental exposure when the optical fiber 108 becomes dislodged. Because the focal point 116 lies within the housing 102, the laser beams 114 will pass through the focal point 116 and dissipate within the housing 102 if the optical fiber 108 is removed. Stated another way, the laser beams 114 beams cannot pass through the aperture 106 unless an optical fiber 108 is positioned within the aperture 106.

In the embodiment depicted in FIG. 1A, axis 110 is perpendicular to the wall 104 defining the aperture 106. Although this approach may be the most efficient to fabricate, one of ordinary skill in the art will readily appreciate that axis 1 10 can be at an angle with respect to wall 104. In most circumstances, axis 1 10 will be substantially coaxial with central axes of aperture 106 and/or optical fiber 108.

Light source 100 can include a single laser 1 12 or a plurality of lasers 1 12. For example, the light source 100 can include between 2 and 150 lasers 112. In one exemplary embodiment constructed by the inventors, light source 100 included 37 lasers 112. However, light source 100 can include other quantities of lasers 112 (e.g., between 1 and 10, between 10 and 20, between 20 and 30, between 30 and 40, between 40 and 50, between 50 and 60, between 60 and 70, between 70 and 80, between 80 and 90, between 90 and 100, between 100 and 1 10, between 110 and 120, between 120 and 130, between 130 and 140, between 140 and 150, and the like).

Lasers 1 12 can be mounted in a mounting plate 118 having a one or more holes 120 oriented at an angle with respect to axis 1 10. Holes 120 can have various angles such that lasers beams 114 generated by lasers 112 mounted within holes 120 each converge on focal point 1 16. For example, holes closer to axis 1 16 may have a smaller angle with respect to axis 116 than holes further from axis 1 16. In one embodiment, holes 120 are arranged in a substantially concentrically circular pattern.

In some embodiments, mounting plate 118 is slidable within housing 102 so that the position of the focal point 116 can be adjusted to reflect various lengths of optical fibers 108. For example, mounting plate 118 can slide along a path defined by slots 122. In some embodiments, the focal point 116 will be between about 15 mm and about 60 mm from the aperture 106. This measurement can be taken from either the internal surface or the external surface of wall 104.

Lasers can be actuated individually, in various subsets, or as a single unit. Actuating individual lasers 1 12 or subsets of lasers 1 12 can be advantageous for several reasons.

First, light entering the optical fiber 108 at relatively small angles with respect to axis 110 will emerge from the working end of the optical fiber 108 at a relatively small angle with respect to an axis defined by the working end of the optical fiber 108 and best illuminate objects that are directly in front of the working end. Likewise, light entering the optical fiber 108 at relatively large angles with respect to axis 110 will emerge from the working end of the optical fiber 108 at a relatively large angle with respect to an axis defined by the working end of the optical fiber 108 and best illuminate objects that are to the side of the working end.

Second, lasers 112 may be configured to emit light at various wavelengths and a user may wish to selectively utilize one or more wavelengths to illuminate one or more objects of interest. For example, in a surgical environment, a healthcare professional may administer a plurality of photosensitive compounds that each bind to a particular type of cell and each have a different excitation frequency. Independent actuation of varying frequencies will allow the healthcare professional to visualize only the desired cells.

Third, the net power output of the light can be modulated as needed.

The particular angles of the lasers 1 12 can be selected based on the parameters (e.g., the diameter) of the optical fiber 108. In general, the angles should be less than the critical angle at which internal reflection within a particular optical fiber will not occur. FIG. IB provides additional views of a light source according to embodiments of the invention. FIG. 1C depicts a side view of mounting plate 118 showing the convergence of laser beams 1 14 on focal point 1 16.

Medical Devices

Referring now to FIG. 2 A, another aspect of the invention provides a medical device 200a including a light source 100 and an instrument 202 including an optical fiber 108. Instrument 202 can be removably coupled to the light source 100 such that the optical fiber 108 lies within an aperture as described above. This allows for the instrument 202 to be removed for disposal or sterilization after a procedure. In one embodiment, the instrument 202 is a laparoscope that can be used for various surgical procedures such as prostatectomies using the nano particle heating techniques described herein.

Instrument 202 can include a camera. Camera can be positioned entirely at the working end 204 of instrument 202 and can include a CCD or CMOS device 206 connected to a monitor and/or recording device 208 by one or more wires 210. Alternatively, camera can be a distributed camera that consists of a lens 206 at the working end 204 coupled to a monitor and/or recording device 208 by one or more fiber optic cables 210. In either form, the camera can be adapted and configured to image at an emission wavelength of a compound of interest that is excited by a wavelength emitted by the light source 100. This adaptation and configuration can be achieved by one or more physical or software filters as will be appreciated by one of skill in the art.

In some embodiments, instrument 202 is adapted and configured for illumination of one or more objects of interest. In other embodiments, instrument 202 is adapted and configured for treatment of one more objects of interest in accordance with known laser medicine and laser surgery techniques. For example, instrument 202 can include one or more lenses 212 adapted and configured to focus the light emitted by the light source into a cutting beam.

Referring now to FIG. 2B, another embodiment of a medical device 200b is depicted. In FIG. 2B and certain other figures, embodiments of the light source described herein are denoted by the LUMINIR™ trademark.

Methods of Illuminating an Object of Interest

Referring now to FIG. 3, a method 300 of illuminating an object of interest is provided. In step S302, a photosensitive compound is administered to a subject. The photosensitive compound can be any compound that responds to one or more wavelengths of light. For example, some compounds fluoresce when exposed to particular wavelengths of light.

In some embodiments, the photosensitive compound preferentially binds to cancerous cells. For example, a photosensitive compound known as YC-27 binds to prostate-specific membrane antigen (PSMA), which is expressed on the surface of prostate cancer cells. YC- 27 is described in publications such as U.S. Patent Application Publication Nos. 201 1/142760 and 2012/009121 and Y. Chen et al, "A low molecular weight PSMA-based fluorescent imaging agent for cancer," 390(3) Biochem. Biophys. Res. Comm. 624-29 (2009).

In other embodiments, the photosensitive compound binds to other cells. For example, a photosensitive compound that binds to nerve cells would allow healthcare professionals to visualize nerves within an organ and avoid damaging these nerves. One exemplary compound, 3,3'-diethylthiatricarbocyanine iodide (DBT), is described C.Wang et al., 31(7) The Journal of Neuroscience 2382-90 (Feb. 16, 2011). Alternatively, an aptamer, a small peptide, or any other known small molecule that specifically binds to nerves (without inhibiting nerve function) could be identified and conjugated with a fluorescent dye.

Other exemplary photosensitive compounds include metal nanoparticles such as iron nanoparticles or gold nanoparticles. Such metal nanoparticles rapidly heat when exposed to near infrared (MR) radiation from a light source as described herein. Heating of metal nanoparticles can increase the sensitivity to adjacent cells to therapeutics.

In step S304, a medical device as described herein applies one or more excitation frequencies to the object of interest. The excitation frequency of interest may vary between photosensitive compounds. For YC-27, the excitation frequency has a wavelength of about 780 nm.

In step S306, the object of interest is optionally imaged at an emission wavelength of the photosensitive compound. For YC-27, the emission wavelength is above 800 nm.

Referring now to FIG. 4, a prototype light source as constructed by the inventors is depicted. The prototype includes two fans to facilitate high air flow and dissipate heat. The use of an aluminum block also facilitates heat dissipation. Multiple toggle switches control the net intensity (power) of the laser output. A weighted swing flap blocks aperture to prevent any stray light from escaping if any of the lasers are out of alignment.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the of the skilled artisan. Such techniques are explained fully in the literature, such as,

"Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989);

"Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987);

"Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

Example 1: Use of LumiNIR to heat iron oxide nanoparticles in vitro

The above-described light source was designed mainly for imaging purposes.

However, it has unexpectedly been discovered that the light source is highly efficacious for the hyperthermal activation of iron oxide nanoparticles. As shown in FIG. 5, the above- described LumiNIR system, which heats at 650nm, 780nm, and 830nm, was able to effectively heat multiple nanoparticles in vitro at 780nm, resulting in a progressive increase in hyperthermal activation over time. The observed data indicate that hyperthermal activation with the LumiNIR system was as effective, if not more effective, that hyperthermal activation with an alternating electromagnetic field.

Example 2: Use of LumiNIR to heat iron oxide nanoparticles in vivo

As the LumiNIR system was effective in heating iron oxide nanoparticles in vitro, it was subsequently tested for its ability to heat iron oxide nanoparticles in vivo. As shown in FIG. 6, three in vivo tissue samples (a skin sample, a first tumor sample, and a second tumor sample) comprising iron oxide nanoparticles were subjected to treatment with the LumiNIR sample in order to induce hyperthermal activation of the iron oxide nanoparticles. Upon activation of the a 5.5 W laser (black arrow, FIG. 6), a pronounced hyperthermal activation was observed for all three samples within the first 500 seconds. To assess the dynamic reactivity of the system, the 5.5 W laser was dimmed in 1.35 W increments (red arrows, FIG. 6) and shown to reduce the heating of the iron oxide nanoparticles over time. The reduction in hyperthermal activation was reversible, as increasing the laser back to 5.5 W (FIG. 6, second black arrow at 3,000 second) resulted in a hyperthermal activation as strong as, if not stronger than, the original activation. Accordingly, the techniques herein provide for a dynamic system with which to provide localized heating of iron oxide nanoparticles either in vitro or in vivo.

It is contemplated within the scope of the disclosure that the compositions and methods described herein may be applied to a variety of therapeutic applications, such as treatment of a variety of neoplasias (e.g., prostate cancer). For example, iron oxide nanoparticles could be injected or delivered into a site positive for prostate cancer. Once delivered, the iron oxide nanoparticles, in combination with the targeted heating methods described above, could be used for a variety of purposes, including, but not limited to: 1) a marker by using localized tissue heating as a thermal marker, e.g., to see if some tumor tissue has been left; 2) a therapy by using localized tissue heating to sensitize the target cells for chemotherapy or radiation. Iron oxide nanoparticles may be readily targeted to a variety of specific target tissues by conjugating them with any of a variety of targeting agents. These applications provide novel uses for hyperthermal activation of iron oxide nanoparticles because existing methods bases on an AMF cannot be used in surgical settings.

It is contemplated within the scope of the disclosure that the above-described system can be used with a variety of nanoparticles amenable to hyperthermal activation, which may readily be identified by one of skill in the art.

OTHER EMBODIMENTS

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. EQUIVALENTS

Although preferred embodiments of the invention have been described using specific terms, such description is for illustrative purposes only, and it is to be understood that changes and variations may be made without departing from the spirit or scope of the following claims.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications, and other references cited herein are hereby expressly incorporated herein in their entireties by reference.

Claims

What is claimed is:

1. A method of heating an iron oxide nanoparticle, the method comprising subjecting the iron oxide nanoparticles to near infrared radiation greater than 650nm.

2. The method of claim 1, where in the near infrared radiation is greater than 680nm.

3. The method of claim 1, where in the near infrared radiation is greater than 780nm.

4. The method of claim 1, where in the near infrared radiation is greater than 830nm.

5. The method of claim 1, where in the near infrared radiation is 680nm.

6. The method of claim 1, where in the near infrared radiation is 780nm.

7. The method of claim 1, where in the near infrared radiation is 830nm.

8. The method of claim 1, wherein the iron oxide nanoparticle is heated using a light source.

9. The method of claim 8, wherein the light source is a LumiNIR light source.

10. The method of claim 8, wherein the light source is laser.

11. The method of claim 1, further comprising targeting the iron oxide nanoparticle to a target tissue.

12. The method of claim 11 , wherein the tissue is selected from the group consisting of a tumor, a breast tissue, a prostate tissue, a skin tissue, and a tumor tissue.

13. The method of claim 1, further comprising administering a photosensitive compound.

14. The method of claim 13, wherein the photosensitive compound responds to one or more wavelengths of light.

15. The method of claim 13, wherein the photosensitive compound is YC-27.

16. A method of treating prostate cancer comprising;

introducing a plurality of iron oxide nanoparticles into prostate cancer cells of a subject; and

hyperthermally activating the iron oxide nanoparticles, thereby treating the prostate cancer of the subject.