WO2016134164A1

WO2016134164A1 - Methods of enhancing cerenkov luminescence using nanoparticles, and compositions related thereto

Info

Publication number: WO2016134164A1
Application number: PCT/US2016/018502
Authority: WO
Inventors: Edwin Pratt; Jan Grimm; Moritz Kircher; Travis M. SHAFFER; Matthew WALL
Original assignee: Memorial Sloan Kettering Cancer Center; Cornell University
Priority date: 2015-02-18
Filing date: 2016-02-18
Publication date: 2016-08-25

Abstract

Cerenkov luminescence occurs when a charged particle travels through a medium faster than a speed of light in that medium. The luminescence generated depends on the refractive index of the material and the energy of the particle traveling through the dielectric medium. Biomedical applications of Cerenkov luminescence currently utilize radionuclides producing Cerenkov light through interactions with tissue, which has a refractive index range of 1.36-1.41. The present disclosure describes the enhancement of Cerenkov luminescence through coupling various radionuclides to different biocompatible nanoparticles that have refractive indices greater than tissue.

Description

METHODS OF ENHANCING CERENKOV LUMINESCENCE USING

NANOP ARTICLES, AND COMPOSITIONS RELATED THERETO

Cross Reference to Related Application

[0001] This application claims the benefit of U.S. Application Serial No. 62/117,928 filed on February 18, 2015, the disclosure of which is hereby incorporated by reference in its entirety.

Field of the Invention

[0002] This invention relates generally to imaging agents and related methods. More particularly, in certain embodiments, the invention relates to radionuclides coupled (covalently or non-covalently) to biocompatible high refractive index nanomaterials for enhanced biomedical imaging and therapeutic applications.

Government Support

[0003] This invention was made with government support under Grant Nos. R01

EB014944-01, R01CA183953, and P30-CA00874 awarded by National Institutes of Health (NIH) and Grant No. DGS 0965983 awarded by the National Science Foundation (NSF). The government has certain rights in this invention.

Background

[0004] Cerenkov luminescence occurs when the velocity of a charged particle exceeds the phase velocity of a medium. The phase velocity is governed in general by Einstein's relativistic kinetic energy equation where phase velocity v, and the speed of light c can be related by the refractive index n such as c/n=v. By increasing the refractive index (RI) of the material and/or environment of a radionuclide, the phase velocity is lowered and charged particles emitted by the radionuclide emit Cerenkov light.

[0005] In a preclinical and/or clinical setting, radionuclides are surrounded by tissues and water which typically have a RI of -1.33-1.41. However, the RI of the environment

surrounding the radionuclide must be greater than about 1.41 in order to increase the yield of emitted particles that produce Cerenkov light. The material properties that govern Cerenkov enhancement remain unknown. Cerenkov enhancement may be produced by altering the RI of the suspension. However, this is not feasible for preclinical and clinical models because serum and tissue become the dominant RI materials, thereby limiting the yield of emitted particles.

[0006] Enhancement of Cerenkov luminescence of radionuclides in joint cartilage detection has been described (U.S. Patent Application No. US 2014/0228682 Al); however, these methods are limited to imaging of cartilage alone. Moreover, the use of radionuclides alone may not fully harness the full effect of enhanced Cerenkov luminescence.

[0007] Nanoparticles are often combined with ionizing radiation sources for in vivo imaging and therapy. These combinations include both radiotracers that emit high-energy photons and beta particles along with proton, photon, and electron beam therapy. Ionizing radiation is routinely used in the clinic and is an area where nanoparticles offer an array of imaging and therapeutic applications. Ionizing radiation sources such as proton, photon, and electron beams or radionuclides are often combined with nanoparticles for various applications.

[0008] In the medical field, nanoparticles have been used as radionuclide platforms for diagnostic purposes such as sentinel lymph node imaging and photodynamic therapy. Other applications outside of in vivo applications include dosimetry instrumentation, and betavoltaic cells on a macro level. However, the lack of understanding of the mechanisms of interaction between ionizing radiation and nanoparticles limit the biomedical applications of radionuclides used in combination with nanoparticles.

[0009] Although the interactions of visible spectrum photons with nanoparticles have been studied, other interactions need to be considered when utilizing ionizing radiation (e.g., high-energy photons or sub-atomic particles) with nanoparticles for improvements in biomedical applications. For example, when a high-energy sub-atomic particle (HEP) is traveling through matter, it dissipates energy until reaching a thermal equilibrium. During this process, the main interaction of the HEP is typically with the electromagnetic field of the atoms rather than direct interaction with the nucleus. For photons, these interactions include 1) photoelectric effect, 2) Compton scattering, 3) coherent scattering, and 4) pair formation. For electrons and positrons, interactions include 1) Cerenkov radiation, 2) excitation, 3) ionization, 4) bremsstrahlung, and 5) positron annihilation.

[0010] The probability of each type of interaction occurring depends on the properties of both the HEP and the matter through which the HEP is interacting. These interactions have facilitated applications, such as: Cerenkov radiation and various nanoparticles for imaging and therapy, quantum dot scintillation under gamma-ray irradiation, and rare-earth nanoparticle excitation by both Cerenkov radiation and gamma-ray irradiation mechanisms. However, there is a need to better understand these interactions for improved biomedical applications in imaging, therapeutics, cell-based assays, etc.

[0011] For medical applications, typically only Cerenkov radiation excitation (e.g., spectrum shifting) or gamma excitation (e.g., radioluminescence) of the nanoparticles are considered as mechanisms. However, other mechanisms of ionizing radiation modulation are possible, and may be beneficial to improved instrumental design, dosimetry calculations, etc. applicable to the medical field.

[0012] There exists a need for high refractive index biocompatible nanomaterials that can deliver radionuclides to sites of interest, produce Cerenkov luminescence for biomedical imaging and therapeutic applications, and exploit the full potential of Cerenkov luminescence of a radionuclide that is absent when using the radionuclide alone.

Summary of the invention

[0013] The present disclosure describes the enhancement of Cerenkov luminescence and alternative interactions of particles by coupling radionuclides (e.g., covalently or non-covalently) to biocompatible nanoparticles that have refractive indices greater than heterogeneous medium (e.g., tissue).

[0014] Described herein are various interactions between high-energy subatomic particles and nanoparticles that result in modulation of the photon flux in both the visible and high-energy spectrum. Furthermore, flux modulation at specific wavelengths is assigned to photoluminescence from visible spectrum photons and excitation and ionization events from high-energy photons and beta particles. These interactions provide an ability to pair

nanoparticles with ionizing radiation sources for use in various biomedical applications (e.g., imaging, therapeutics, cell-based assays, etc.).

[0015] Also described herein are interactions between ionizing radiation (e.g., including electrons, positrons, and high-energy photons) and nanoparticles with an array of compositions. Both high-energy and visible photon modulations are investigated with (i) high-energy photon emitter ^mTc, (ii) low energy pure beta emitters S and P, and radionuclides that emit both low energy betas and high-energy photons ¹⁸F, ⁶⁸Ga, ⁹⁰Y, and ¹⁷⁷Lu.

[0016] Described herein are simple systems, for example, silica (S P) and germanium

(GeNP) nanoparticles, that do not exhibit photoluminescence. Also described herein are more complex systems, for example, titanium dioxide (Ti0₂) and hafnium dioxide (Hf0₂), that do exhibit photoluminescence. Also described are systems that exhibit photoluminescence and scintillation from various HEPs: for example, europium oxide nanoparticles (Eu₂0₃). Each system reveals mechanisms of photon modulation and are applied to applications in vitro and in vivo.

[0017] In one aspect, the invention is directed to a method of imaging tissue (e.g., a luminescence detection method), which method comprises administering one or more agents to the tissue (e.g., in vitro, or administering to a subject, in vivo) so that the tissue (e.g., the subject) is receiving a nanoparticle and/or liposome having one or more radionuclides either coupled thereto (e.g., covalently or non-covalently coupled, e.g., with or without a chelator, e.g., with or without a linker moiety between the radionuclide and the nanoparticle) or within its vicinity (e.g., within 3 millimeters, e.g., within 2 millimeters in water, e.g., within 1 millimeter in tissue, e.g., within a Cerenkov distance) in the tissue (e.g., the tissue of the subject) the nanoparticle or liposome and the one or more radionuclides; and detecting luminescence produced in the tissue (e.g., of the subject) as a result of the administered nanoparticle or liposome and one or more radionuclides (e.g., wherein the nanoparticle or liposome has higher refractive index than the tissue, e.g., the tissue of the subject in the vicinity of the administered one or more agents being imaged). [0018] In certain embodiments, the one or more radionuclides comprises one or more members selected from the set consisting of ⁸⁹Zr, ⁹⁰Y, ⁶⁴Cu, ¹⁷⁷Lu, ⁶⁸Ga, ¹⁸FDG, ³³P, ³⁵S, ¹⁴C, 3H, ¹⁸F, and combinations thereof.

[0019] In certain embodiments, the one or more radionuclides comprises ³³P. In certain embodiments, the one or more radionuclides comprises ³⁵S.

[0020] In certain embodiments, the nanoparticle and/or liposome has diameter no greater than about 250 nm (e.g., no greater than 200 nm, no greater than 150 nm, no greater than 140 nm, or no greater than 100 nm). In certain embodiments, the nanoparticle is a member selected from the group consisting of silica, aluminum oxide, titanium dioxide, zinc oxide, quartz, cuprite, titania (rutile), titania (anatase), europium, hafnium, gadolinium oxide, and diamond.

[0021] In certain embodiments, the one or more agents exhibits greater radiance/ μθ than a solution (e.g., buffer solution) having the same one or more radionuclide, but without the nanoparticle (or liposome). In certain embodiments, the one or more agents has at least 1.5 times the radiance^Ci of the solution of the radionuclide alone (e.g., at least 2.0 times, at least 2.5 times, or at least 3.0 times, at least 20 times, at least 40 times, at least 80 times, at least 100 times, at least 500 times, at least 1000 times, at least 1500 times, at least 2000 times). In certain embodiments, the one or more agents exhibits an average radiance of greater than 8 x 10⁵ p/s/cm²/sr (e.g., at least 1.5 times the average radiance of a buffer solution of the radionuclide without the nanoparticle and/or liposome, e.g., at least 1.6 times greater, e.g., at least 1.63 times greater, e.g., at least 2.0 times greater, e.g., for ⁹⁰Y) (e.g., at least 10 times the average radiance of a buffer solution of the radionuclide without the nanoparticle and/or liposome, e.g., at least 20 times greater, e.g., at least 100 times greater, e.g., at least 2000 times greater, e.g., for ³⁵S) [0022] In certain embodiments, the one or more agents exhibits at least 0.75 times the average radiance of a buffer solution of the radionuclide without the nanoparticle and/or liposome, e.g., at least 0.5 times greater, e.g., at least 0.2 times greater, e.g., at least 0.1 times greater, e.g., for GaAs or GaP nanoparticles).

[0023] In certain embodiments, the nanoparticle and/or liposome comprise targeting ligands to target a tumor. In certain embodiments, the tissue is tumor tissue.

[0024] In another aspect, the invention is directed to a composition of matter comprising a nanoparticle and/or liposome having one or more radionuclides either coupled thereto (e.g., covalently or non-covalently coupled, e.g., with or without a chelator, e.g., with or without a linker moiety between the radionuclide and the nanoparticle) or within its vicinity (e.g., within 3 millimeters, e.g., within 2 millimeters in water, e.g., within 1 millimeter in tissue, e.g., within a Cerenkov distance), the nanoparticle and/or liposome having a refractive index of at least 1.2 (e.g., at least 1.45, at least 1.49, at least 1.7, at least 1.9, at least 2.0, at least 2.4, or at least 2.5) such that the composition exhibits enhanced Cerenkov luminescence.

[0025] In certain embodiments, the one or more radionuclides comprises one or more members selected from the set consisting of ⁸⁹Zr, ⁹⁰Y, ⁶⁴Cu, ¹⁷⁷Lu, ⁶⁸Ga, ¹⁸FDG, ³³P, ³⁵S, ¹⁴C, ³H, ¹⁸F, and combinations thereof.

[0026] In certain embodiments, the one or more radionuclides comprises ³³P. In certain embodiments, the one or more radionuclides comprises ³⁵S.

[0027] In certain embodiments, the nanoparticle and/or liposome has diameter no greater than about 250 nm (e.g., no greater than 200 nm, no greater than 150 nm, no greater than 140 nm, or no greater than 100 nm). In certain embodiments, the composition comprises a nanoparticle and the nanoparticle is a member selected from the group consisting of silica, aluminum oxide, titanium dioxide, zinc oxide, quartz, cuprite, titania (rutile), titania (anatase), europium, hafnium, gadolinium oxide, and diamond.

[0028] In certain embodiments, the composition exhibits greater radiance/ μθ than a solution (e.g., buffer solution) having the same radionuclide as the composition, but without the nanoparticle (or liposome).

[0029] In certain embodiments, the composition has at least 1.5 times the radiance^Ci of the solution of the radionuclide alone (e.g., at least 2.0 times, at least 2.5 times, or at least 3.0 times, at least 20 times, at least 40 times, at least 80 times, at least 100 times, at least 500 times, at least 1000 times, at least 1500 times, at least 2000 times).

[0030] In certain embodiments, the composition exhibits an average radiance of greater than 8 x 10⁵ p/s/cm²/sr (e.g., at least 1.5 times the average radiance of a buffer solution of the radionuclide without the nanoparticle or liposome, e.g., at least 1.6 times greater, e.g., at least 1.63 times greater, e.g., at least 2.0 times greater, e.g., for ⁹⁰Y) (e.g., at least 10 times the average radiance of a buffer solution of the radionuclide without the nanoparticle or liposome, e.g., at least 20 times greater, e.g., at least 100 times greater, e.g., at least 2000 times greater, e.g., for ³⁵S)

[0031] In certain embodiments, the composition exhibits at least 0.75 times the average radiance of a buffer solution of the radionuclide without the nanoparticle or liposome, e.g., at least 0.5 times greater, e.g., at least 0.2 times greater, e.g., at least 0.1 times greater, e.g., for GaAs or GaP nanoparticles).

[0032] In certain embodiments, the radionuclide emits low energy beta minus particles and does not emit detectable gamma and/or x-ray particles (e.g., wherein the radionuclide is

33 35 14 3

selected from the group consisting of P, S, C, and H). [0033] In another aspect, the invention is directed to a method of detecting a radionuclide, the method comprising: administering (e.g., spraying) one or more agents comprising a nanoparticle and/or liposome to a surface; and detecting (e.g., via a camera) luminescence produced by the nanoparticle and/or liposome having one or more radionuclides either coupled thereto (e.g., covalently or non-covalently coupled, e.g., with or without a chelator, e.g., with or without a linker moiety between the radionuclide and the nanoparticle) or within its vicinity (e.g., within 3 millimeters, e.g., within 2 millimeters in water, e.g., within 1 millimeter in tissue, e.g., within a Cerenkov distance), (e.g., wherein the radionuclide emits low energy beta minus particles and does not emit detectable gamma and/or x-ray particles (e.g.,

33 35 14 3

wherein the radionuclide is selected from the group consisting of P, S, C, and H)).

[0034] In certain embodiments, the method comprises administering the one or more agents to the surface, thereby resulting in the composition.

[0035] In another aspect, the invention is directed to a method for detecting biological processes (e.g., amino acids, phosphate, e.g., in vitro), the method comprising tethering (e.g., via

33 35 14 3

click-chemistry, etc.) low energy radionuclides (e.g., P, S, C, and/or H) that do not emit detectable gamma and/or x-ray particles to amino acids (e.g., a ³⁵S-Cysteine or ³⁵S-methionine) or phosphates (e.g., a ³³P-phosphate) to form a tethered radionuclide composition; contacting (e.g., bringing together within a Cerenkov distance) a nanoparticle and the tethered radionuclide composition (e.g., contacting the tethered radionuclide composition with nanoparticles in solution, e.g., Ε¾0₃ nanoparticles, e.g., wherein the solution has a concentration of 1E10, 5E10, 1E11, 2E11, 5E11, 1E12 nanoparticles/mL, e.g., wherein the nanoparticle is coated on a surface); and detecting luminescence resulting from the contacting of the nanoparticle and the tethered radionuclide composition (e.g., via a high sensitivity camera). [0036] In certain embodiments, contacting the nanoparticle and the tethered radionuclide composition results in the composition.

Definitions

[0037] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0038] In this application, the use of "or" means "and/or" unless stated otherwise. As used in this application, the term "comprise" and variations of the term, such as "comprising" and "comprises," are not intended to exclude other additives, components, integers or steps. As used in this application, the terms "about" and "approximately" are used as equivalents. Any numerals used in this application with or without about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art. In certain

embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%), 2%), 1%), or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0039] "Agent" or "One or more agents": The term "agent" may include nanoparticles, radionuclides, conjugates of nanoparticles and radionuclides, carriers, excipients, etc.

[0040] "Administration ": The term "administration" refers to introducing a substance into a subject. In general, any route of administration may be utilized including, for example, parenteral (e.g., intravenous), oral, topical, subcutaneous, peritoneal, intraarterial, inhalation, vaginal, rectal, nasal, introduction into the cerebrospinal fluid, or instillation into body compartments. In certain embodiments, administration is oral. Additionally or alternatively, in certain embodiments, administration is parenteral. In certain embodiments, administration is intravenous.

[0041] "Biocompatible": The term "biocompatible", as used herein is intended to describe materials that do not elicit a substantial detrimental response in vivo. In certain embodiments, the materials are "biocompatible" if they are not toxic to cells. In certain embodiments, materials are "biocompatible" if their addition to cells in vitro results in less than or equal to 20% cell death, and/or their administration in vivo does not induce inflammation or other such adverse effects. In certain embodiments, materials are biodegradable.

[0042] "Biodegradable" . As used herein, "biodegradable" materials are those that, when introduced into cells, are broken down by cellular machinery {e.g., enzymatic degradation) or by hydrolysis into components that cells can either reuse or dispose of without significant toxic effects on the cells. In certain embodiments, components generated by breakdown of a biodegradable material do not induce inflammation and/or other adverse effects in vivo. In certain embodiments, biodegradable materials are enzymatically broken down. Alternatively or additionally, in certain embodiments, biodegradable materials are broken down by hydrolysis. In certain embodiments, biodegradable polymeric materials break down into their component polymers. In certain embodiments, breakdown of biodegradable materials (including, for example, biodegradable polymeric materials) includes hydrolysis of ester bonds. In certain embodiments, breakdown of materials (including, for example, biodegradable polymeric materials) includes cleavage of urethane linkages. [0043] "Carrier": As used herein, "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

[0044] "Detector": As used herein, the term "detector" includes any detector of electromagnetic radiation including, but not limited to, CCD camera, photomultiplier tubes, photodiodes, and avalanche photodiodes. In certain embodiments, the detector is a germanium detector.

[0045] "Sensor": As used herein, the term "sensor" includes any sensor of

electromagnetic radiation including, but not limited to, CCD camera, photomultiplier tubes, photodiodes, and avalanche photodiodes, unless otherwise evident from the context.

[0046] "Substantially": As used herein, the term "substantially", and grammatic equivalents, refer to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the art will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.

[0047] "Subject": As used herein, the term "subject" includes humans and mammals

(e.g., mice, rats, pigs, cats, dogs, and horses). In many embodiments, subjects are be mammals, particularly primates, especially humans. In certain embodiments, subjects are livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. In certain embodiments (e.g., particularly in research contexts) subject mammals will be , for example, rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine such as inbred pigs and the like.

[0048] "Therapeutic agent": As used herein, the phrase "therapeutic agent" refers to any agent that has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect, when administered to a subject.

[0049] "Treatment": As used herein, the term "treatment" (also "treat" or "treating") refers to any administration of a substance that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition. Such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In certain

embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In certain embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition.

[0050] Drawings are presented herein for illustration purposes, not for limitation.

Brief description of drawings

[0051] FIG. 1 depicts that the majority of ¹⁸F positrons (shaded) do not reach the

Cerenkov threshold in water, while the majority of ⁹⁰ Y beta particles do. [0052] FIG. 2 depicts a facile radiolabeling method to stably attach radioisotopes to certain metal-oxide nanoparticles.

[0053] FIG. 3 A shows Cerenkov luminescence of ⁹⁰Y at various wavelengths.

[0054] FIG. 3B depicts Cerenkov Enhancement over buffer of various high refractive index nanoparticles.

[0055] FIG. 4A depicts Cerenkov Enhancement of ⁹⁰Y using various high refractive index nanoparticles using an open emission filter (IVIS instrumentation).

[0056] FIG. 4B depicts Cerenkov Enhancement of silica nanoparticles and their absorbance within the wavelengths of 500-800 nm.

[0057] FIG. 4C depicts ⁹⁰Y Enhancement with nanoparticles normalized to signal MES buffer (as shown in FIG. 4A).

[0058] FIG. 5 A shows Cerenkov enhancement of ⁶⁴Cu using liposomes. Luminescence values plotted comparing buffer with activity to liposomes extruded and stored in the same buffer.

[0059] FIG. 5B shows Cerenkov enhancement of ⁶⁸Ga using liposomes. Luminescence values plotted comparing buffer with activity to liposomes extruded and stored in the same buffer.

[0060] FIG. 5C shows Cerenkov luminescence of ⁹⁰Y using liposomes. Luminescence values plotted comparing buffer with activity to liposomes extruded and stored in the same buffer.

[0061] FIG. 6 shows the relative Cerenkov luminescence enhancement of ⁶⁴Cu, ⁶⁸Ga, and

⁹⁰Y using liposomes. [0062] FIG. 7 shows a plot of RMS distance spread calculated as described in Mitchell et al. and interpolated for various beta-emitting nuclides such as ⁶⁴Cu, ⁸⁹Zr and ⁶⁸Ga.

[0063] FIG. 8 shows Cerenkov enhancement of ⁶⁴ Cu, ⁶⁸Ga, and ⁹⁰Y using liposomes normalized to the RMS spread distance of the beta particle. Copper which has a low beta particle kinetic energy compared to that of Yttrium experiences a greater enhancement in part to the increase in refractive index.

[0064] FIG. 9A shows a schematic of seeded growth of a silica nanoparticle using a modified Stober process outlined by Bogush. Seeded growth occurs via tetra-ethyl-orthosilicate (TEOS) + H20 precursors.

[0065] FIG. 9B shows radiance per activity for MES buffer with activity, a radiolabeled silicon nanoparticle (Si P), and a radiolabeled Si P that was seeded. The particle size change was from 48.3±2.5nm to 68.1±1.8nm. Enhancement of seeded radiolabeled SiNP was 1.5X compared to MES buffer and 1.2X compared to smaller radiolabeled SiNP.

[0066] FIGS. 10A and 10B show average radiance ((p/s/cm²/sr)xl0⁴) for ⁹⁰Y, ⁶⁸Ga, ⁶⁴Cu,

18 89 177

FDG, °¾ and ¹ "Lu versus becquerel (Bq) (10A) and ratio of average radiance/activity concentration ((p/s/cm²/sr)/kBq^L)) versus ⁹⁰Y, ⁶⁸Ga, ⁶⁴Cu, ¹⁸FDG, ⁸⁹Zr, and ¹⁷⁷Lu.

[0067] FIG. 11 depicts β- Cerenkov luminescence radionuclides and MeV spectra with a

Cerenkov threshold in tissue (-0.2 MeV) for ⁶⁴Cu, ¹⁸F, ⁸⁹Zr, ⁹⁰Y, ⁶⁸Ga, ¹⁷⁷Lu, ³³P, and ³⁵S.

[0068] FIGS. 12A - 12D also show that the refractive index and absorbance properties of a nanoparticle can vary with wavelength and are nanoparticle dependent. Without wishing to be bound to any theory, in certain embodiments, transparency affects Cerenkov luminescence.

[0069] FIG. 13 shows ¹⁷⁷Lu fold enhancement to water per particles per mL for various types of nanoparticles. "#5" refers to 10 nm and "#6" refers to 30 nm. [0070] FIG. 14 shows larger enhancement to water per particles per mL for various types of nanoparticles by replacing ¹⁷⁷Lu (FIG. 13) with ³⁵S

[0071] FIG. 15 shows a plot of various nanoparticle enhancements to water compared to refractive index.

[0072] FIGS. 16A and 16B show radiance (p/s) for silica nanoparticles (S P) and germanium (Ge) using different radionuclides.

[0073] FIG. 17 shows time dependence of measurement in imaged corrected for radioactive decay.

[0074] FIG. 18 shows ¹⁷⁷Lu nanoparticle enhancement to water.

[0075] FIG. 19 shows ³⁵ S nanoparticle enhancement to water.

[0076] FIGS. 20A and 20B show increased enhancement for ³⁵S, although total radiance with EU2O3 varies by 4X (at 620 nm) from ⁹⁰Y, which is the highest energy radionuclide of the radionuclides used.

[0077] FIGS. 21A and 21B show IVIS spectra of 1E12 nanoparticles 30μϋϊ ⁶⁸Ga.

[0078] FIG. 21 A shows Cerenkov luminescence contribution of flat enhancement spectra for various nanoparticles, water, and glucose.

[0079] FIG. 2 IB shows EU2O3 or yttrium aluminate nanopowder (YAG) and Gd0₃ atomic transition peaks.

[0080] FIG. 22 shows different energies for ^99mTC and glucose at peaks (x-rays: 18.2,

18.3, 20.5, 20.6 keV; gammas: 140 keV).

[0081] FIG. 23 shows a ^99mTC and Eu₂0₃ four previously unidentified characteristic peaks at 40.80 keV, 41.56 keV, 47.00 keV, and 48.2 keV. [0082] FIGS. 24 and 25 shows peaks for ^{i / 7}Lu (x-rays: 54.6, 55.7, 62.9, 63.2, and 64.9 keV; gammas: 71.6. 112.9, and 208 keV) and Gd₂0₃. (e.g., four previously unidentified peaks with lower energy than Eu₂0₃ from ³⁵S).

[0083] FIG. 24 shows ¹⁷⁷Lu Ge detector overlay with glucose (dotted line) and Gd₂0₃

(solid line).

[0084] FIG. 25 shows comparison of ¹⁷⁷Lu with Gd₂0₃ and Eu₂0₃ showing shift in four peaks based upon nanoparticle added while preserving radionuclide signature energy.

[0085] FIG. 26 shows peaks for ¹⁸FDG and Eu₂0₃ with radioactivity peak at 511 keV.

[0086] FIG. 27 shows peaks for ⁶⁸Ga and Eu₂0₃ with radioactivity peak at 511 keV.

[0087] FIG. 28 shows peaks for ³⁵S and Eu₂0₃. No x-rays or gammas are seen from ³⁵S alone. Addition of Eu₂0₃ yields quartet of peaks in the 40 keV region.

[0088] FIG. 29 shows 1 μϋϊ and 30 μϋϊ detection limits for ³⁵ S in Ε„2θ₃ and Hf0₂ respectively. Increasing activity levels leads to stronger luminescence signals as sdescribbed herein. Note that ³⁵S cannot be optically imaged in H₂0 or Glucose alone.

[0089] FIG. 30 shows Hf0₂ particle titration with a fixed 30 μθ of activity per well.

Subsequent time points show a change in the nanoparticle radiance curve that reflects a nonlinear relationship at different "dark" time points (0 min, 5 min, 10 min, 15 min), where dark is minutes in IVIS box away from ambient light.

[0090] FIG. 31 depicts in vivo data showing that the light emitted by 18FDG

radionuclides is enhanced by 1.5 times when Hf0₂ nanoparticles are co-injected compared to radionuclide alone. Matrigel and Matrigel/Hf0₂ injections served as negative controls. In certain embodiments, this technique has applications in improved optical sensitivity of tumor regions. [0091] FIG. 32 shows photoluminescence of 1E12 nanoparticles in 1 mL of glucose.

Exposure was 300 seconds Fl, where Fl is a lens aperature that is fully open (where light in equals light out).

[0092] FIG. 33 shows ³⁵S luminescence enhancements change with time in IVIS. This represents the effect of photoluminescence on enhancement factor at low radiance radionuclides.

[0093] FIG. 34 shows an exemplary radioluminescence setup containing 24 well plate with 1 mL of nanoparticles (1), black paper (2), plexi glass (3), and 24 well plate containing 30 μθ of a particular radionuclide in H₂0 (4).

[0094] FIG. 35 shows enhancement of Hf0₂ and Ge nanoparticles using 30 μθ of ³³P with 1E11 particles per mL (maximum concentration).

[0095] FIG. 36 shows same data as shown in FIG. 35; however, FIG. 36 shows radiance

(p/s) (compared to enhancement as provided in FIG. 35).

Detailed Description

[0096] It is contemplated that methods of the claimed invention encompass variations and adaptations developed using information from the embodiments described herein.

[0097] Throughout the description, where compositions are described as having, including, or comprising specific components, or where methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are

compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are methods according to the present invention that consist essentially of, or consist of, the recited processing steps. [0098] It should be understood that the order of steps or order for performing certain action is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0099] It should be understood that the order of steps or order for performing certain action is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0100] The mention herein of any publication, for example, in the Background section, is not an admission that the publication serves as prior art with respect to any of the claims presented herein. The Background section is presented for purposes of clarity and is not meant as a description of prior art with respect to any claim.

[0101] The present disclosure describes the enhancement of Cerenkov luminescence and alternative interactions of particles by coupling radionuclides (e.g., covalently or non-covalently) to biocompatible nanoparticles that have refractive indices greater than heterogeneous medium (e.g., tissue).

[0102] Described herein are various interactions between high-energy subatomic particles and nanoparticles that result in modulation of the photon flux in both the visible and high-energy spectrum. Furthermore, flux modulation at specific wavelengths is assigned to photoluminescence from visible spectrum photons and excitation and ionization events from high-energy photons and beta particles. These interactions provide an ability to pair

[0103] Also described herein are interactions between ionizing radiation (e.g., including electrons, positrons, and high-energy photons) and nanoparticles with an array of compositions. Both high-energy and visible photon modulations are investigated with (i) high-energy photon emitter ^99mTc, (ii) low energy pure beta emitters ³⁵ S and ³²P, and radionuclides that emit both low energy betas and high-energy photons ¹⁸F, ⁶⁸Ga, ⁹⁰Y, and ¹⁷⁷Lu.

[0104] Described herein are simple systems, for example, silica (S P) and germanium

(Ge P) nanoparticles, that do not exhibit photoluminescence. Also described herein are more complex systems, for example, titanium dioxide (Ti0₂) and hafnium dioxide (Hf0₂), that do exhibit photoluminescence. Also described are systems that exhibit photoluminescence and scintillation from various HEPs: for example, europium oxide nanoparticles (Eu₂0₃). Each system reveals mechanisms of photon modulation and are applied to applications in vitro and in vivo.

[0105] Embodiments described herein exhibit enhanced Cerenkov luminescence through the use of high refractive index nanoparticles. Cerenkov luminescence occurs when a charged particle travels through a medium faster than a speed of light. The luminescence generated depends on the refractive index (RI) of the material and the energy of the particle traveling through the dielectric medium. Biomedical applications of Cerenkov luminescence presently utilize radionuclides producing Cerenkov light through interactions with tissue, which has a refractive index range of about 1.33-1.41.

[0106] In certain embodiments, the enhancement of Cerenkov luminescence in a biological environment is seen to result from coupling radionuclides to bio-compatible nanoparticles which have refractive indices higher than tissue. In certain embodiments, radionuclides can provide enhancement ranging from about 25% to 250% when coupled with high refractive index nanoparticles relative to samples in buffer alone. In certain embodiments, any biocompatible nanoparticle with a refractive index greater than that of tissue can provide enhancement, although other factors (e.g., kinetic energy of the beta particle, the optical density of the particle, absorbance and scattering of the nanoparticles, etc.) may also affect the enhancement.

[0107] The amount of Cerenkov luminescence is mostly dependent on the refractive index (RI) of the medium as expressed by the Frank-Tamm equation (Equation 1), with the refractive index defined as n. The RI of a substance, or medium, describes how light propagates through the medium (Equation 2).

^ = photons emitted per distance

a = fine structure constant (1/137)

β = velocity of charged particle relative to speed of light in medium

n = refractive index of medium

λ₁ λ₂ = wavelengths of interest c

n =— (Equation 2)

v

n = refractive index of medium

c = speed of light in a vacuum

v = speed of light in medium

[0108] The relativistic kinetic energy of emitted particles (such as positrons and electrons from beta decay) can be calculated:

E = mc² (Equation 3)

[0109] By increasing the refractive index of the material directly surrounding the radionuclide, a greater proportion of Cerenkov radiation can be generated. Beta particles have a range dependent on the radionuclide and medium of interaction, with typical ranges of millimeters before annihilation. Despite this range, the beta particle will have a decreasing kinetic energy as it travels through each successive medium. The Frank-Tamm equation as shown in Equation 1 describes the output of the Cerenkov luminescence as a function of distance and highlights that particles with lower kinetic energy, hence lower speed of light ratio, will emit less light in a given refractive index medium.

[0110] Consistent with Equation 1 is the finding that, in order to maintain a similar yield of light over any distance, an increase in refractive index, n, will allow a proportional increase in particle velocity relative to the speed of light. For high-energy beta emitters such as ⁶⁸Ga and ⁹⁰ Y, the kinetic energy threshold is exceeded for the majority of the beta particles emitted. In contrast, ¹⁸F and ⁶⁴Cu, with a lower kinetic energy distribution of beta particles, produces fewer photons per emitted particle as depicted in FIG. 1.

[0111] In certain embodiments, methods and features (e.g., radiolabelling of

nanoparticles, optimization of nanoparticles stability) described in Shaffer et al., Nano letters 2015: 15(2): 864-8., which is incorporated herein by reference in its entirety, can be used.

[0112] In certain embodiments, beta particles themselves (e.g., not gamma and/or x-rays) lead to strong enhancement of an optical signature. In certain embodiments, the nanoparticles used in the present disclosure include, but are not limited to, rare earth particles, ZnO, Hf()₂, Si₃N₄, Dy₂0₃, Ti0₂ anatase, Ti0₂ rutile, A1₂0₃, Ge, TiN, A1₂0₃, GaAs, GaP, Eu₂0₃, etc.

[0113] In certain embodiments, ³³P and ³⁵S are used in biomedical research in combination with Eu₂0₃ to yield Cerenkov enhancement. The enhancement is a result from interactions between subatomic particles and nanoparticles. In certain embodiments, the addition of these radionuclides (e.g., P and S) into amino acids or phosphate groups enable enhanced optical readout for plate based assays upon adding a nanoparticle solution.

[0114] In certain embodiments, Eu₂0₃ produces a strong total photo flux. Hu et al.

("Optical imaging of articular cartilage degeneration using near-infrared dipicolylamine probes," Biomaterials 35(2014) 7511-7521) published on Eu₂0₃ nanoparticles. However, Hu et al.

attributed the luminescence properties of Eu₂0₃ nanoparticles to the gamma and x-ray

interaction. As described herein, the accurate interaction is clearly though ³⁵S, as ³⁵S does not produce gammas or x-rays (e.g., alone in H₂0 or glucose). As described herein, Eu₂0₃ and 30 μθ of ³⁵S yield a luminescence enhancement of nearly 2500X that of ³⁵S in water (FIG. 14).

[0115] Combining radioisotope and nanoparticles for in vitro and/or in vivo use provides high payload and multimodal possibilities (e.g., dual mode imaging of Cerenkov and magnetic resonance). The visible light emanating from beta-emitting radiotracers has allowed pre-clinical and clinical luminescence imaging, but the photon flux is quite low. Here, nanoparticles are shown to modulate the Cerenkov signal through various mechanisms for use in medical applications. For example, interactions between the nanoparticles and radionuclides are used in PET imaging reconstruction. As the stopping power for a positron is strongly dependent on the atomic (Z) number of surrounding matter, higher Z numbers lead to shorter positron ranges, which benefits in vivo PET imaging reconstruction. In certain embodiments, the present disclosure provides methods for lymph node imaging, tumor imaging, and alternative in vivo applications that vary based upon the concentration of nanoparticles used.

[0116] In certain embodiments, nanoparticles are sprayed on a surface to detect radiation.

For example, ³⁵ S and ³³P are currently challenging to detect on available machines due to their low energy levels (e.g., lack positron and gamma particles). The present disclosure provides the ability to detect radionuclides more easily. Examples of other radionuclides that are beta minus emitting (and no gamma and/or x-ray generation) include but are not limited to ¹⁴C and ³H.

[0117] In certain embodiments, rare earth nanoparticles (e.g., gadolinium) are able to be detected with magnetic resonance. In certain embodiments, the present disclosure provides dual- mode detection (e.g., optical and MR detection). In certain embodiments, reactive oxygen species are generated with the nanoparticles (e.g., europium) to induce oxidative and/or catalytic properties.

Examples

High refractive index nanoparticles

[0118] Si0₂ particles were made as described by Shaffer et al. Ti0₂, A1₂0₃, and ZnO were purchased as less than 150 nm dispersions from Sigma Aldrich, Item #'s 700347, 702129, and 721085 respectively. The refractive indexes described in the literature are listed in Table 1. In certain embodiments, other high refractive index and biocompatible nanoparticles not listed in Table 1 may be used. The optical density of silica nanoparticles with a diameter of 140 nm is as measured in FIG. 4.

[0119] Table 1 shows exemplary high refractive index nanoparticles at a wavelength of

500 nm. Table 1 shows refractive indexes for nanoparticles of different sizes and, in certain embodiments, different refractive indexes for the same material of nanoparticle. For example, Ti0₂ includes a refractive index material for anatase and rutile Ti0₂ and different sizes. Note that the listed refractive index is a bulk measurement at 500 nm.

Table 1

Cerenkov enhancement using radionuclide bound high refractive index nanoparticles

[0120] Silica nanoparticles, as described by Shaffer et al., were used as chelator free constructs to probe Cerenkov enhancement. FIG. 2 shows the method for chelator-free radiolabeling of nanoparticles. This method can also be successfully applied for radiolabeling high refractive index nanoparticles (e.g., aluminum oxide nanoparticles, titanium dioxide nanoparticles). As shown in FIGS. 3A and 3B, radionuclides tested with all of the nanoparticles include ⁸⁹Zr and ⁹⁰Y. Silica nanoparticle enhancement was further tested with ⁶⁴Cu and ¹⁷⁷Lu. Compared to the activity in buffer (e.g., a refractive index of 1.33), attaching the radionuclide to nanoparticles with high refractive indexes (e.g., greater than 1.49) resulted in enhanced Cerenkov luminescence in accordance with theory. As shown in FIG. 4A, the degree of Cerenkov

Enhancement of ⁹⁰Y depends on the selected nanoparticle. Moreover, Cerenkov Enhancement of silica nanoparticles and their absorbance are stable over the visible spectrum (e.g, within the wavelengths of 500 nm - 800 nm) as depicted in FIG. 4B.

Cerenkov enhancement using radionuclide bound liposomes

[0121] In addition to high refractive index solid cores, liposomes were also tested for

Cerenkov enhancement given their shell like structure. Liposomes were prepared by solvent casting a lipid film of DSPE-PEG, DSPC, and Cholesterol (5/62/33 mol percent) and rehydrated with 1M HEPES buffer with gentle bath sonication. Liposomes were then extruded at 55°C through 1 μπι, 0.2 μπι and then 0.1 μπι filters ten times each to achieve liposomes with an average size of 220 nm. These liposomes were then incubated with ⁶⁴Cu, ⁶⁸Ga, and ⁹⁰Y at a total volume of 100 μΕ with lOOuCi of activity per sample. Samples and measurements were done in triplicate, with the exception of the ⁹⁰Y HEPES buffer ,which has a n=l . Luminescence values were acquired in phantom wells over 1000 ms to quantify the Cerenkov enhancement relative to the same activity in buffer as depicted in FIGS. 5A-5C.

Liposome enhancement relative to buffer by nuclide

[0122] As shown in FIG. 6, ⁹⁰Y shows the largest enhancement and is also the highest energy emitting beta particle. Higher kinetic energy beta particles travel over a greater spread distance before annihilation than lower kinetic energy beta particles. Using the root means square (RMS) spread of a beta particle published by Mitchell et al. versus the beta particle simulated distance, a coarse measurement of enhancement per area can be estimated. The trend of beta particle energy and RMS distance as described by Mitchell et al. can be used to interpolate additional RMS values for intermediate nuclides as depicted in FIG. 7. [0123] The enhancement per nuclide based on the change in refractive index can be estimated when the enhancement of various nuclides with liposomes is normalized by the enhancement by the nuclide beta emitting RMS travel distance. FIG. 8 depicts confirmation of the diagram (Mitchell et al.) reproduced in FIG. 1. FIG. 8 shows that nuclides with low/threshold beta particle energy for Cerenkov, such as ⁶⁴Cu, will see the largest enhancement by increasing the refractive index.

[0124] Table 2 summarizes values of average radiance for ⁹⁰Y, comparing values observed for the ⁹⁰Y buffer solution (without nanoparticles) to values observed with ⁹⁰Y coupled to silica nanoparticles (1.60 times greater average radiance), coupled to aluminum oxide nanoparticles (1.63 times greater average radiance), and coupled to titanium dioxide nanoparticles (2.23 times greater average radiance).

[0125] Table 2 shows average radiance values for ⁹⁰Y coupled to high refractive index nanoparticles.

Table 2 yttrium-90:

Avg Radiance [p/s/cm²/sr]

~ioo _μα

Buffer (RI 1.33) (control): 5.41E+05

silica nanoparticles (RI 1.49): 8.63E+05

aluminum oxide (RI 1.76): 8.84E+05

titanium dioxide (RI-2.5): 1.21E+06 [0126] Table 3 shows characteristic x-ray spectra from NIST x-ray transitions database.

KL refers to the transition between K shell and L shell, KM refers to the transition between K shell and M shell, and KN refers to the transition between K shell and N shell.

Table 3

[0127] FIGS. 9A and 9B depict that a radionuclide can be in a volume (e.g., the radionuclide does not have to be attached) with a nanoparticle. For example, the nanoparticle is near a positron and/or a β^" particle.

[0128] FIG. 9A shows a schematic of seeded growth of a silica nanoparticle using modified Stober process outlined by Bogush. Seeded growth occurs via TEOS + H20 precursors.

[0129] FIG. 9B shows radiance per activity for MES buffer with activity, a radiolabeled silicon nanoparticle (SiNP), and a radiolabeled SiNP that was seeded. The particle size change was from 48.3±2.5nm to 68.1±1.8nm. Enhancement of seeded radiolabeled SiNP was 1.5X compared to MES buffer and 1.2X compared to smaller radiolabeled SiNP. [0130] In certain embodiments, the radionuclide does not have to be attached to the particle. In certain embodiments, the radionuclide is within an interaction volume in which the radionuclide decays. By full width half maximum, range varies between 0 mm - 1mm in tissue while in water this expands to nearly 2 mm (not shown). These distances highlight relative proximity of the radionuclide and nanoparticle for the beta interaction to make a contribution.

[0131] FIGS. 10A and 10B show average radiance ((p/s/cm²/sr)xl0⁴) for ⁹⁰Y, ⁶⁸Ga, ⁶⁴Cu,

18 89 177

FDG, °¾ and ¹ "Lu versus becquerel (Bq) (10A) and ratio of average radiance/activity concentration ((p/s/cm²/sr)/kBq^L)) versus ⁹⁰Y, ⁶⁸Ga, ⁶⁴Cu, ¹⁸FDG, ⁸⁹Zr, and ¹⁷⁷Lu. As shown in FIGS. 10A and 10B, ⁹⁰ Y associated with the nanoparticles produces the most light and ¹⁷⁷Lu associated with the nanoparticles produces the least amount of light.

[0132] FIG. 11 depicts β- Cerenkov luminescence radionuclides MeV spectra with a

Cerenkov threshold in tissue (-0.2 MeV) for ⁶⁴Cu, ¹⁸F, ⁸⁹Zr, ⁹⁰Y, ⁶⁸Ga, ¹⁷⁷Lu, ³³P, and ³⁵S. The area under the curve represents the Cerenkov luminescence threshold in tissue. Prior to the present disclosure, isotopes with no gamma and/or positron particles and with low levels of β^", such as ³³P and ³⁵S, were not able to be optically imaged. Advantages to imaging isotopes with low levels of β^" include but are not limited to the following: (i) longer half-life of low energy isotopes (e.g., about 85 days or years (e.g., ¹⁴H, ³H)) provides for longer trajectory imaging studies and (ii) such isotopes can be naturally found in various biological processes. For example, ³⁵ S can be incorporated into cysteine amino acids or ³³P can be used to image phosphorous. Using the present disclosure, for example, a cysteine amino acid or phosphorous can be imaged in cell-based assays if a nanoparticle is in close proximity to the amino acid and/or phosphorus. In certain embodiments, a nanoparticle-coated plate is used to image low levels of β^" from radionuclides, such as ³³P and ³⁵S, in cell based assays. [0133] FIGS. 12A - 12D also show that the refractive index and absorbance properties of a nanoparticle can vary with wavelength and are nanoparticle dependent. Without wishing to be bound to any theory, in certain embodiments, transparency affects Cerenkov luminescence (e.g., enhancement of light or absorption of light).

[0134] FIG. 13 shows ¹⁷⁷Lu fold enhancement to water per particles per mL for various types of nanoparticles. In certain embodiments, a higher concentration of nanoparticles produces more light. In certain embodiments, a high concentration of nanoparticle absorbs the light (e.g., serves as a light quencher). In certain embodiments, targeting agents are added to the nanoparticles and used to target tumors in vitro and/or in vivo.

[0135] FIG. 14 shows larger enhancement to water per particles per mL for various types of nanoparticles by replacing ¹⁷⁷Lu (FIG. 13) with ³⁵S. For example, the less energy that the nanoparticle, the greater enhancement that is measured.

[0136] FIG. 15 shows a plot of various nanoparticle enhancements to water compared to refractive index. FIG. 15 shows that multiple factors, e.g., not just refractive index, contribute to fold-enhancement of light.

[0137] FIGS. 16A and 16B show radiance (p/s) for silica nanoparticles (S P) and germanium (Ge) using different radionuclides. Silica nanoparticles do not seem to enhance light at lel2 particles/mL; however, enhancement is seen for germanium at this concentration.

[0138] FIG. 17 shows time dependence of measurement in imaged corrected for radioactive decay.

[0139] FIG. 18 shows ¹⁷⁷Lu nanoparticle enhancement to water.

[0140] FIG. 19 shows ³⁵S nanoparticle enhancement to water. [0141] FIGS. 20A and 20B show increased enhancement for S, although total radiance with EU2O3 varies by 4X less (at 620 nm) from ⁹⁰Y, which is the highest energy radionuclide of the radionuclides used, although ³⁵ S has 10X less energy than ⁹⁰ Y. Accordingly, more light per energy is measured when using ³⁵S. Note that EU2O3 has only been shown with ¹⁸FDG, ⁶⁸Ga and ^99mTc radionuclides (e.g., radionuclides that are not β emitting).

[0142] FIGS. 21 A and 21B show IVIS spectra of 1E12 nanoparticles per mL at 30μϋϊ

⁶⁸Ga.

[0143] FIG. 21A shows Cerenkov luminescence contribution of flat enhancement spectra for various nanoparticles, water, and glucose. FIG. 21 A shows that these nanoparticles have no spectral dependence.

[0144] FIG. 2 IB shows Eu₂0₃ or YAG and Gd0₃ atomic transition peaks. FIG. 2 IB shows that these nanoparticles have a spectral dependence based on interactions other than Cerenkov luminescence (e.g., properties other than refractive index). For example, alternative interactions may include gamma and/or x-ray interactions between the radionuclide and nanoparticle.

[0145] FIG. 22 shows different energies for ^99mTC and glucose at peaks (x-rays: 18.2,

18.3, 20.5, 20.6 keV; gammas: 140 keV).

[0146] FIG. 23 shows Eu₂0₃ characteristic four peaks and demonstrates that the radionuclide does not have to be attached to the nanoparticle to see the effect. In certain embodiments, material characterization is performed via this type of spectroscopy.

[0147] FIGS. 24 and 25 shows peaks for ¹⁷⁷Lu (x-rays: 54.6, 55.7, 62.9, 63.2, and 64.9 keV; gammas: 71.6. 112.9, and 208 keV) and Gd₂0₃. (e.g., four new peaks with lower energy than Eu₂0₃ from ³⁵S). FIGS. 24 and 25 show that the peaks are characteristic to the nanoparticle regardless of which radionuclide is used. For example, the characteristic four peaks of Eu₂0₃ exhibit the same energy regardless of the radionuclide associated with the nanoparticle. This spectroscopy technique provides the ability to parse out the nanoparticle being used (e.g., Gd₂0₃ or Eu₂0₃). The measurements shown in FIG. 25 are beta minus, with high energy gamma, or high energy gamma itself.

[0148] FIG. 26 shows peaks for ¹⁸FDG and Eu₂0₃ with radioactivity peak at 511 keV.

[0149] FIG. 27 shows peaks for ⁶⁸Ga and Eu₂0₃ with radioactivity peak at 511 keV.

[0150] FIG. 28 shows peaks for ³⁵S and Eu₂0₃. No x-rays or gammas are seen from ³⁵S alone. Addition of Eu₂0₃ yields quartet of peaks in the 40 keV region. No x-rays or gammas are seen from ³⁵S alone. Addition of Eu₂0₃ yields quartet of peaks in the 40 keV region.

Accordingly, without wishing to be bound to any theory, the beta-minus particle or the positron is contributing to the light enhancement.

[0151] ^99mTc results show characteristic spectra in rare earths as seen in both beta emitting radionuclides. Intensity of characteristic x-rays not calibrated, but appear to be more in line with abundance using β^" radionuclides.

[0152] FIG. 29 shows a ³⁵S activity titration of Eu₂0₃ and Hf0₂ nanoparticles at lei 1 particles per mL from a range of 0.5 μθ to 50 μθ.

[0153] FIG. 30 shows that enhancement from nanoparticle concentration is not linear at different "dark" time points (0 min, 5 min, 10 min, 15 min), where dark is minutes in IVIS box away from ambient light. The nanoparticles were titrated at 30 μθ per well.

[0154] FIG. 31 depicts in vivo data showing that the light emitted by 18FDG

radionuclides is enhanced by 1.5 times when Hf0₂ nanoparticles are co-injected compared to radionuclide alone. Matrigel and Matrigel/Hf0₂ injections served as negative controls. In certain embodiments, this technique has applications in improved optical sensitivity of tumor regions.

[0155] FIG. 32 shows photoluminescence of 1E12 nanoparticles in 1 mL of glucose.

Exposure was 300 seconds F l, where Fl is a lens aperature that is fully open (where light in equals light out).

[0156] FIG. 33 shows ³⁵S luminescence enhancements change with time in IVIS. This represents the effect of photoluminescence on enhancement factor at low radiance radionuclides.

[0157] FIG. 34 shows an exemplary radioluminescence setup containing 24 well plate with 1 mL of nanoparticles (1), black paper (2), plexi glass (3), and 24 well plate containing 30 μθ of a particular radionuclide in H₂0 (4).

[0158] FIG. 35 shows enhancement of Hf0₂ and Ge nanoparticles using 30 μθ of ³³P with 1E1 1 particles per mL (maximum concentration).

[0159] FIG. 36 shows same data as shown in FIG. 35; however, FIG. 36 shows radiance

(p/s) (compared to enhancement as provided in FIG. 35).

Total luminescence measurements

[0160] Using the plate design and nanoparticles concentrations as described below, 30iC of radioactivity was added per well and imaged on the IVIS Spectrum. Subsequent scans using the open filter were done to track the loss in total luminescence with time. Radiance values were decay corrected and thus losses represent the photoluminescence contribution.

Spectrum scans to assess the wavelength output in the 500 nm - 840 nm region were also done on the IVIS in 20 nm increments, open filter (FIG. 32). For more energetic radionuclides such as ¹⁸FDG, scans of 2 minutes F l were used instead. For the most energetic radionuclides like ⁹⁰Y exposure times of 30 s - 60 s were used. [0161] Applications include, but are not limited to, tethering low energy radionuclides that produce no visible light to amino acids (e.g., ³⁵S Cysteine or methionine) or phosphates (e.g., ³³P). Accumulation of amino acid or phosphate can then be optically measured by adding a fixed amount of Eu₂0₃ nanoparticles in solution. In certain embodiments, the particles are also coated on a plate. Assay readout can be performed using a high sensitivity camera like that is USED by the IVIS Spectrum. Activity correlates with luminescence.

Radioluminescence

[0162] Radioluminescence studies were conducted in a similar manner (FIGS. 33 and 34) to the luminescence studies, but with the addition of a plexi glass, paper cover and the radioactivity placed at the bottom in a separate 24 well plate. This design removes the Cerenkov luminescence and beta interactions by the radionuclide and retains the gamma and x-ray interactions if possible. While this design does not account for identical geometry for all well (e.g., some wells will see more activity based on location in plate), this tool serves as a good rank order mechanism, along with a follow on ^99mTc study which represents gamma excitation only. Activity and nanoparticle titration

[0163] To test the effect of nanoparticle concentration, 6 concentrations of nanoparticles were tested (e.g., 1E10, 5E10, 1E11, 2E11, 5E11, 1E12 nanoparticles / mL) for Hf0₂ with 30 μθ ⁸⁹Zr. Doubling of particles can lead to a less than linear response in increased radiance, though concentrations below 1E11 nanoparticles per mL appear linear as shown in FIG. 30. The activity titration ranges from 0.5 to 50 μθ per well using a fixed 1E11 particles per mL of either Hf0₂ or Eu₂0₃. A clear optical signal at 1 μθ is shown in FIG. 30 with ³⁵S. This previously undetected optical signal enables Cerenkov imaging of ³⁵S. Furthermore, without wishing to be bound to any theory, pure beta emitters are harder to measure than positron or gamma emitting radionuclides, suggesting that nanoparticle sprays with Eu₂0₃ can be used with a camera to measure amounts of ³⁵S on a surface. For example 1 μθ of ³⁵S cannot be seen optically, nor by a Geiger counter or x-ray or gamma detector. As described herein, by adding Eu₂0₃, the 1 μθ of ³⁵S can be imaged and quantified, as shown in FIG. 30. Furthermore, in certain embodiments monitoring of ³⁵S is detected in a Ge detector if the sample in question and the nanoparticles are combined and placed in the detector.

Germanium (Ge) detector

[0164] The Ge detector is designed to measure the high energy x-ray and gamma rays given off during radionuclide decay. Every radionuclide has a signature energy pattern that is used to determine the radionuclide. Upon adding nanoparticles to the detector with activity, nanoparticles (e.g., rare earth nanoparticles) produced four previously unidentified peaks in the energy spectrum, without observable loss in existing known radionuclide peaks. The peak energies are dependent on the type of nanoparticle, while the abundance of the peak depends on the type of the radionuclide used. The produced peak energies correspond to characteristic x-ray energies, enabling material identification through radionuclide addition. Positrons produced the weakest new peaks, while SPECT nuclides such as ^99mTc produced higher abundance peaks. Beta minus emitting radionuclides (e.g., ¹⁷⁷Lu, ³⁵S) produced the strongest peak signals, though still a minority peak by counts. Geometry was not taken into account for this study so integrated values are not available. The Canberra Ge detector was run for 2 hours containing 1 mL of nanoparticles (1E12 particles) and radionuclide. Radionuclide amounts varied from 3 iCi^' of ⁹⁰Y to 100 μθ of ³⁵S. Amounts were adjusted to maintain between 1% and 5% dead time on the detector, which is related to count rate (FIGS. 22-28). [0165] Applications can be directed towards characterization of rare earth elements by addition of radionuclide to sample. In certain embodiments, any beta minus emitter is used to identify the element it is mixed with, as long as the element or mineral has characteristic x-rays that are known, in the low x-ray range, and do no overlap with the spectral signature of the nuclide.

In vivo imaging using ionizing radiation interactions with nanoparticles

[0166] Matrigel plugs were prepared via mixture of radioisotope, matrigel, and 2 nM

(50μΙ.) (or 1E12 particles per mL) of nanoparticle solution in an eppendorf tube. 100 μΐ_^ of the solution (25 μθ) was injected subcutaneously and both PET and IVIS optical imaging were conducted. A control matrigel plug with no nanoparticles (e.g., only matrigel, ¹⁸FDG, and saline) was used as a comparison (FIG. 31).

[0167] Prostate tumor models were prepared by injecting 1.5 x 10⁶ 22RV1 cells (ATCC) subcutaneously in the left flank. Three weeks after xenografts injection, mice were fasted for 4 hours followed by intravenous ¹⁸FDG injection (400 μθ). At 1 hour post-injection, PET and IVIS imaging was conducted. Immediately following IVIS imaging, 2 nM (50μΙ.) (or 1E12 particles per mL)) of nanoparticle solution was applied topically to the matrigel and the mouse was reimaged. The solution was then removed and the mouse was imaged a third time. ⁶⁸Ga- PSMA (600 μθ) was injected intravenously and PET imaging conducted 2 hours post-injection. IVIS imaging and topical application of P was conducted as described for ¹⁸FDG.

[0168] Lymph node imaging was conducted via injection of E₂0₃ (10 μΕ, 2 nM or 1E12 particles per mL) into the left footpad and a saline control (10 μΕ) into the right footpad, followed by injection of radionuclide. PET and IVIS imaging was conducted at 1 hour and 4 hours post-injection. P luminescence and enhancement

[0169] 1 mL of nanoparticles in solution was applied per well. Concentrations of OF02 and Ge were titrated at lower levels, with 1E11 nanoparticles per mL as the maximum concentration (e.g., as compared to 1E12 nanoaprticles per mL in ³⁵S experiments described herein). Enahncement was calculated by comparing to H₂0 luminescence, as described herein. Exposure was 300 seconds Fl . FIG. 35 shows enhancement of Hf0₂ and Ge nanoparticles using 30 iCi^' of ³³P with 1E11 particles per mL (maximum concentration). FIG. 36 shows same data as shown in FIG. 35; however, FIG. 36 shows radiance (p/s) (compared to enhancement as provided in FIG. 35).

Nanoparticle preparation

[0170] The majority of the nanoparticles were purchased from either American Elements or Sigma Aldrich with the exception of synthesized silica nanoparticles. Nanopowders were suspended in 60% by weight glucose with the aid of tip sonication to create a monodisperse nanoparticle suspension. Silica nanoparticles were prepared via a modified Stober method.

[0171] Suspension of the nanoparticles in 60% by weight glucose was achieved by sonication in a 500W tip or cup sonicator. Sonication was done to disperse the nanoparticles and minimize sedimentation or aggregation over the course of the experiment. Absorbance measurements were taken to correct for size measurements by DLS. Refractive index of the nanomaterial was assumed for the DLS measurements. Transmission electron microscopy of identical nanoparticles was done (from an aqueous dilution) on a Jeol electron microscope to confirm particle size and morphology. Nanosight measurements were done to confirm the ballpark concentration of the nanosuspensions. Particle concentrations were set at 1E10, 1E11, and 1E12 particles per mL in 60% Glucose. Nanoparticle characterization (SI Absorbance, Photoluminescence)

[0172] Nanoparticle morphology and diameter were determined using both transmission electron microscopy (TEM) and dynamic light scattering (DLS). A Nanosight instrument was used to determine particle concentration. The optical refractive index of the nanoparticles was assumed to be a bulk state refractive index. The absorbance and photo-luminescence was determined via cuvette measurements of 1E10 particles per mL or lower and extrapolated when exceeding 1 optical density (OD).

[0173] Nanoparticles at 1E10, 1E11, and 1E12 (IX) particles per mL in 60% glucose were added to a black walled 24 well plate. Plate design includes triplicate wells of H₂0, Glucose, and three concentration levels of NPs for two different NPs. Particles were stored under ambient fluorescent light for more than 15 minutes before measurement in the IVIS spectrum. Samples were imaged with an open filter at Fl for 300s per exposure as seen in the figure below. Subsequent exposures of particles retained in the IVIS Spectrum, which is a dark light sealed imaging chamber, showed the loss of total luminescence with time.

Photoluminescence decay curves could be determined to confirm. Losses varied by particle while some (YAG, Gd₂0₃) had increases in luminescence likely due to settling of the nanosuspension, thus altering the absorption of the 1 mL dispersion. These values represent no addition of radioactivity.

Radiotracer production (SI)

35 33 177 90

[0174] S, P, Lu, and Y were purchased from PerkinElmer as liquid solutions.

When appropriate, solutions were brought to neutral pH prior to addition to nanoparticle solutions. ⁸⁹Zr was produced on the Memorial Sloan Kettering Cancer Center cyclotron and purified as zirconium oxalate. Zirconium oxalate was neutralized with sodium carbonate prior to use. Cu was produced on a cyclotron at Washington University in Saint Louis and eluted as copper chloride in 0.05 N HC1. ⁶⁸Ga was produced on a germanium generator and eluted in 0.3 N HC1. This was neutralized with 28% ammonium hydroxide prior to use.

Gamma and x-ray interactions for visible light modulation

[0175] 24 well black walled plates were used for luminescence imaging. Nanoparticle solutions were prepared in 60% glucose by weight with 1E10, 1E11 and 1E12 particles per mL. ImL of nanosuspension was diluted with 5 μΐ. of the radionuclide of interest, targeting 30 μθ per well at time of measurement. Enhancement was normalized to the total flux of the radionuclide in H₂0.

High-energy photon measurements

[0176] The radioisotope of interest was placed in 1 mL H₂0 in a 24 well black walled plate upon which a poly(methyl methacrylate) plate and black paper was then placed. On top of the black paper another 24 well plate containing in triplicate wells of H₂0, 50% glucose, or nanoparticle solution. IVIS imaging was conducted with an open filter. ROIs were drawn over the wells and background subtracted.

Beta particle interactions for visible light modulation

[0177] ³⁵S in H₂0 was prepared in 1 mL volumes ranging from 0.5-30 μθί in 24 well black-walled plates. ³⁵S was added to either H₂0, 60% glucose in H₂0, or P (HO or EO). IVIS imaging was conducted for 5 minutes (binning 8, fstop 1, XYZ). ROIs were drawn over each well and radiance (photons) values were determined.

Claims

What is claimed is:

1. A method of imaging tissue (e.g., a luminescence detection method), which method comprises

administering one or more agents to the tissue (e.g., in vitro, or administering to a subject, in vivo) so that the tissue (e.g., the subject) is receiving a nanoparticle and/or liposome having one or more radionuclides either coupled thereto (e.g., covalently or non-covalently coupled, e.g., with or without a chelator, e.g., with or without a linker moiety between the radionuclide and the nanoparticle) or within its vicinity (e.g., within 3 millimeters, e.g., within 2 millimeters in water, e.g., within 1 millimeter in tissue, e.g., within a Cerenkov distance) in the tissue (e.g., the tissue of the subject) the nanoparticle or liposome and the one or more radionuclides; and detecting luminescence produced in the tissue (e.g., of the subject) as a result of the administered nanoparticle or liposome and one or more radionuclides (e.g., wherein the nanoparticle or liposome has higher refractive index than the tissue, e.g., the tissue of the subject in the vicinity of the administered one or more agents being imaged).

2. The method of claim 1, wherein the one or more radionuclides comprises one or more members selected from the set consisting of ⁸⁹Zr, ⁹⁰Y, ⁶⁴Cu, ¹⁷⁷Lu, ⁶⁸Ga, ¹⁸FDG, ³³P, ³⁵S, ¹⁴C, ³H, ¹⁸F, and combinations thereof.

3. The method of any one of the preceding claims, wherein the one or more radionuclides comprises ³³P.

4. The method of any one of the preceding claims, wherein the one or more radionuclides comprises ³⁵S.

5. The method of any one of the preceding claims, wherein the nanoparticle and/or liposome has diameter no greater than about 250 nm (e.g., no greater than 200 nm, no greater than 150 nm, no greater than 140 nm, or no greater than 100 nm).

6. The method of any one of the preceding claims, wherein the nanoparticle is a member selected from the group consisting of silica, aluminum oxide, titanium dioxide, zinc oxide, quartz, cuprite, titania (rutile), titania (anatase), europium, hafnium, gadolinium oxide, and diamond.

7. The method of any one of the preceding claims, wherein the one or more agents exhibits greater radiance^Ci than a solution (e.g., buffer solution) having the same one or more radionuclide, but without the nanoparticle (or liposome).

8. The method of claim 7, wherein the one or more agents has at least 1.5 times the radiance^Ci of the solution of the radionuclide alone (e.g., at least 2.0 times, at least 2.5 times, or at least 3.0 times, at least 20 times, at least 40 times, at least 80 times, at least 100 times, at least 500 times, at least 1000 times, at least 1500 times, at least 2000 times).

9. The method of claim 7, wherein the one or more agents exhibits an average radiance of greater than 8 x 10⁵ p/s/cm²/sr (e.g., at least 1.5 times the average radiance of a buffer solution of the radionuclide without the nanoparticle and/or liposome, e.g., at least 1.6 times greater, e.g., at least 1.63 times greater, e.g., at least 2.0 times greater, e.g., for ⁹⁰Y) (e.g., at least 10 times the average radiance of a buffer solution of the radionuclide without the nanoparticle and/or liposome, e.g., at least 20 times greater, e.g., at least 100 times greater, e.g., at least 2000 times greater, e.g., for ³⁵S)

10. The method of claim 7, wherein the one or more agents exhibits at least 0.75 times the average radiance of a buffer solution of the radionuclide without the nanoparticle and/or liposome, e.g., at least 0.5 times greater, e.g., at least 0.2 times greater, e.g., at least 0.1 times greater, e.g., for GaAs or GaP nanoparticles).

11. The method of any one of the preceding claims, wherein the nanoparticle and/or liposome comprise targeting ligands to target a tumor.

12. The method of any one of the preceding claims, wherein the tissue is tumor tissue.

13. A composition of matter comprising a nanoparticle and/or liposome having one or more radionuclides either coupled thereto (e.g., covalently or non-covalently coupled, e.g., with or without a chelator, e.g., with or without a linker moiety between the radionuclide and the nanoparticle) or within its vicinity (e.g., within 3 millimeters, e.g., within 2 millimeters in water, e.g., within 1 millimeter in tissue, e.g., within a Cerenkov distance), the nanoparticle and/or liposome having a refractive index of at least 1.2 (e.g., at least 1.45, at least 1.49, at least 1.7, at least 1.9, at least 2.0, at least 2.4, or at least 2.5) such that the composition exhibits enhanced Cerenkov luminescence.

14. The composition of claim 13, wherein the one or more radionuclides comprises one or more members selected from the set consisting of ⁸⁹Zr, ⁹⁰Y, ⁶⁴Cu, ¹⁷⁷Lu, ⁶⁸Ga, ¹⁸FDG, ³³P, ³⁵S, ¹⁴C, ³H, ¹⁸F, and combinations thereof.

15. The composition of claims 13 or 14, wherein the one or more radionuclides comprises ³³P.

16. The composition of any one of claims 13 to 15, wherein the one or more radionuclides comprises ³⁵S.

17. The composition of any one of claims 13 to 16, wherein the nanoparticle and/or liposome has diameter no greater than about 250 nm (e.g., no greater than 200 nm, no greater than 150 nm, no greater than 140 nm, or no greater than 100 nm).

18. The composition of any one of claims 13 to 17, wherein the composition comprises a nanoparticle and the nanoparticle is a member selected from the group consisting of silica, aluminum oxide, titanium dioxide, zinc oxide, quartz, cuprite, titania (rutile), titania (anatase), europium, hafnium, gadolinium oxide, and diamond.

19. The composition of any one of claims 13 to 18, wherein the composition exhibits greater radiance^Ci than a solution (e.g., buffer solution) having the same radionuclide as the composition, but without the nanoparticle (or liposome).

20. The composition of claim 19, wherein the composition has at least 1.5 times the radiance^Ci of the solution of the radionuclide alone (e.g., at least 2.0 times, at least 2.5 times, or at least 3.0 times, at least 20 times, at least 40 times, at least 80 times, at least 100 times, at least 500 times, at least 1000 times, at least 1500 times, at least 2000 times).

21. The composition of any one claims 13 to 20, wherein the composition exhibits an average radiance of greater than 8 x 10⁵ p/s/cm²/sr (e.g., at least 1.5 times the average radiance of a buffer solution of the radionuclide without the nanoparticle or liposome, e.g., at least 1.6 times greater, e.g., at least 1.63 times greater, e.g., at least 2.0 times greater, e.g., for ⁹⁰Y) (e.g., at least 10 times the average radiance of a buffer solution of the radionuclide without the nanoparticle or liposome, e.g., at least 20 times greater, e.g., at least 100 times greater, e.g., at least 2000 times greater, e.g., for ³⁵S)

22. The composition of any one of claims 13 to 21, wherein the composition exhibits at least 0.75 times the average radiance of a buffer solution of the radionuclide without the nanoparticle or liposome, e.g., at least 0.5 times greater, e.g., at least 0.2 times greater, e.g., at least 0.1 times greater, e.g., for GaAs or GaP nanoparticles).

23. The composition of any one of claims 13 to 22, wherein the radionuclide emits low energy beta minus particles and does not emit detectable gamma and/or x-ray particles (e.g.,

33 35 14 3

wherein the radionuclide is selected from the group consisting of P, S, C, and H).

24. A method of detecting a radionuclide, the method comprising:

administering (e.g., spraying) one or more agents comprising a nanoparticle and/or liposome to a surface; and

detecting (e.g., via a camera) luminescence produced by the nanoparticle and/or liposome having one or more radionuclides either coupled thereto (e.g., covalently or non-covalently coupled, e.g., with or without a chelator, e.g., with or without a linker moiety between the radionuclide and the nanoparticle) or within its vicinity (e.g., within 3 millimeters, e.g., within 2 millimeters in water, e.g., within 1 millimeter in tissue, e.g., within a Cerenkov distance), (e.g., wherein the radionuclide emits low energy beta minus particles and does not emit detectable gamma and/or x-ray particles (e.g., wherein the radionuclide is selected from the group consisting of ³³P, ³⁵S, ¹⁴C, and ³H)).

25. The method of claim 24, comprising:

administering the one or more agents to the surface, thereby resulting in the composition of any one of claims 13 to 23.

26. A method for detecting biological processes (e.g., amino acids, phosphate, e.g., in vitro), the method comprising: tethering (e.g., via click-chemistry, etc.) low energy radionuclides (e.g., P, S, C, and/or ³H) that do not emit detectable gamma and/or x-ray particles to amino acids (e.g., a ³⁵S- Cysteine or ³⁵S-methionine) or phosphates (e.g., a ³³P-phosphate) to form a tethered radionuclide composition;

contacting (e.g., bringing together within a Cerenkov distance) a nanoparticle and the tethered radionuclide composition (e.g., contacting the tethered radionuclide composition with nanoparticles in solution, e.g., Ε¾0₃ nanoparticles, e.g., wherein the solution has a concentration of 1E10, 5E10, 1E11, 2E11, 5E11, 1E12 nanoparticles/mL, e.g., wherein the nanoparticle is coated on a surface); and

detecting luminescence resulting from the contacting of the nanoparticle and the tethered radionuclide composition (e.g., via a high sensitivity camera).

27. The method of claim 26, wherein contacting the nanoparticle and the tethered

radionuclide composition results in the composition of any one of claims 13 to 23.