# US 10,307,527 B2 · Protein-based nanobubble x-ray imaging method and agent > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,307,527 B2 - Document ID: US10307527B2 - Status: Granted - Kind: B2 - Application number: 14/692,075 - Filed: 2015-04-21 - Published: 2015-10-22 - Granted: 2019-06-04 - Earliest claimed: 2014-04-21 - Assignees: Brown University; Montana State University - Applicants: Brown University; Montana State University - Inventors: Christoph Rose-Petruck; Trevor Douglas; Danielle Rand; Masaki Uchida - Web page: https://patentgrep.com/patent/US10307527B2 ## CPC - A61M 5/007: {for contrast media} - A61B 6/481: {involving the use of contrast agents} - A61K 49/0461: {Dispersions, colloids, emulsions or suspensions} - A61B 6/06: Diaphragms - A61M: DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY (introducing media into or onto the bodies of animals A61D7/00; means for inserting tampons A61F13/26; devices for administering food or medicines orally A61J; containers for collecting, storing or administering blood or medical fluids A61J1/05); DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY (surgery A61B; chemical aspects of surgical articles A61L); DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR {(Electrotherapy, e.g. producing anaesthesia by the use of alternating or intermittent currents A61N1/36021)} - A61B: DIAGNOSIS; SURGERY; IDENTIFICATION (usefulness limited to only animals A61D) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract Systems, compositions, methods and kits employ protein shells, such as ferritin or viral capsid shells, herein called nanobubbles, to enhance X-ray images of cells or body tissue under certain x-ray imaging methods. The nanobubbles have sub-micron size such as about 10 nm, about 40, 60, or 100 nm and may be functionalized for effective delivery to or uptake by a target tissue, in vivo or a cell culture. The nanobubbles are hollow, having a central core which may be empty or contain a fluid, such that the shells effectively form long-lived bubbles in the imaged environment, and are of low electron density and have different scattering properties than the surrounding tissue. X-ray imaging by spatial frequency heterodyne imaging enhances visualization or detection of tissue regions bearing the shells. The protein shells may be further treated to assure biocompatibility and/or to resist undesired interactions with non-targeted tissue, such as scavenging by the liver, or attack by the immune system. For example the nanobubbles may be filled with a hydrophobic gas such as perfluoropropane (octafluoropropane) to maintain their shape and resist hydrophilic interactions and delay the degradation of the protein shells. The nanobubbles may also be coated or treated by a surface-functionalization processes to effectively target specific tissues or tumor types, allow parenteral delivery, and/or deliver drugs or other agents to the imaged sites—so that when visualized by X-ray scatter imaging the presence of the nanobubbles indicates the existence and extent of the tumor or diseased tissue. ## Claims ### Claim 1 A method of imaging cells or a tissue, the method comprising: providing to the cells or tissue, nanoparticles formed of protein, the nanoparticles having an outer diameter under about 60 nm disposed around a particle interior, the particle interior containing a hydrophobic material trapped or residing therein that prevents entry of aqueous material and thereby enhances stability of the nanoparticles in vivo, wherein the nanoparticles lack metal or other high Z coating, component or material that would otherwise increase x-ray transmission imaging contrast, irradiating the cells or the tissue with an X-ray source; and, X-ray scatter imaging the cells or the tissue with spatial frequency harmonic heterodyne image processing to effectively images regions of tissue or cells bearing the nano particles. ### Claim 2 (depends on 1) The method according to claim 1, wherein the nanoparticles are viral protein shells. ### Claim 3 (depends on 1) The method according to claim 1, wherein the nanoparticles are synthetic protein shells produced by recombinant production of a protein and self-assembly into shell structures. ### Claim 4 (depends on 1) The method according to claim 1, wherein the nanoparticles include protein shells comprising a virion coat protein or peptide. ### Claim 5 (depends on 4) The method according to claim 4, wherein the nanoparticles are protein shells that comprise a virion selected from the group consisting of: prokaryotic, protozoan, eukaryotic, virus-like particles, and bacteriophage virions, for example, wherein the virus shells are obtained from one selected from the group consisting of: Poxviridae, Entomopoxyviridae, Baculoviridae, Eubaculovirinae, Nudibaculovirinae, Polydnaviridae, Ichnovirus, Iridoviridae, Bracovirus, Parvoviridae, Flavivirdae, Tagviridae, Bunyaviridae, Rhabdoviridae, Reoviridae, Bimaviridae, Picornaviridae, Tetraviridae and Nadoviridae. ### Claim 6 (depends on 4) The method according to claim 4, wherein the nanoparticle is a protein nanoshell that is semi-synthetic, is a synthetic peptide, or is a recombinantly produced protein. ### Claim 7 (depends on 1) The method according to claim 1, wherein the nanoparticles further comprise at least one material selected from the group consisting of: a polymer, a binding agent, and an organic material. ### Claim 8 (depends on 1) The method according to claim 1, wherein the hydrophobic material is or includes a hydrophobic gas such as perfluoropropane that excludes entry of aqueous. ### Claim 9 (depends on 1) The method according to claim 1, wherein irradiating the sample comprises locating or inserting an absorption grid adjacent to the sample between an X-ray source and a detector, and/or wherein imaging includes the step of positioning a grid between the sample and a detector and performing spatial frequency heterodyne image processing to selectively enhance regions of scattering by protein nanoparticles. ### Claim 10 (depends on 1) The method according to claim 1, wherein the nanoparticles comprise a binding agent that specifically binds the cells or the tissue, wherein the binding agent comprises at least one selected from the group of: a drug, a protein, a carbohydrate, and a nucleotide sequence. ### Claim 11 (depends on 10) The method according to claim 10, applied to tissue which comprises a plurality of cells in order to image or detect targeted cells selected from at least one of the group of: cancerous, non-cancerous, epithelial, hematopoietic, stem, spleen, kidney, pancreas, prostate, liver, neuron, breast, glial, muscle, sperm, heart, lung, ocular, brain, bone marrow, fetal, blood, leukocyte, and lymphocyte. ### Claim 12 (depends on 10) The method according to claim 10, applied to a tumor or cancer tissue selected from the group consisting of: melanoma; colon carcinoma; pancreatic; lymphoma; glioma; lung; esophagus; mammary; prostate; head; neck; ovarian; stomach; kidney; liver; and hepatocellular carcinoma. ### Claim 13 (depends on 11) The method according to claim 11, wherein the step of providing is performed by administering the nanoparticles parenterally. ### Claim 14 (depends on 11) The method according to claim 11, wherein the step of providing is performed by at least one route selected from the group consisting of: intra-tumoral, intravenous, intra-ocular, intra-muscular, subcutaneous, and intraperitoneal and oral. ### Claim 15 (depends on 1) The method according to claim 1 wherein the nanoparticles further include at least one fluid or contrast agent selected from the group consisting of: a magnetic resonance imaging (MRI) agent, a positive contrast agent, or a negative contrast agent, or, for example, an oil, a metal ion (e.g., iron and magnesium), or a metal compound. ### Claim 16 A kit for imaging cells or a tissue, the kit comprising: a plurality of nanoparticles that bind to and/or are taken up by the cells or the tissue, wherein the nanoparticles comprise a protein nano shell filled with a low electron density fluid and operable to function as empty protein nanoparticles visible with enhanced contrast by scatter X-ray imaging, the X-ray imaging with spatial frequency harmonic heterodyne image processing to effectively images regions of tissue or cells bearing the nano particles, the nanoparticles having an outer diameter under about 60 nm disposed around a particle interior, the particle interior containing a hydrophobic material trapped or residing therein that prevents entry of aqueous material and thereby enhances stability of the nanoparticles in vivo, wherein the nanoparticles lack metal or other high Z coating, component or material that would otherwise increase X-ray transmission imaging contrast. ### Claim 17 (depends on 16) The kit according to claim 16, wherein the protein shell comprises a viral protein shell or a protein shell, such as a virion coat protein or peptide, and optionally surrounds or is embedded in an exterior surface of at least one nanoparticle. ### Claim 18 (depends on 17) The kit according to claim 17, wherein the shell comprises a virion selected from the group consisting of: prokaryotic, protozoan, eukaryotic, virus-like particles, and bacteriophage virions or the shell is obtained from one selected from the group consisting of: Poxviridae, Entomopoxyviridae, Baculoviridae, Eubaculovirinae, Nudibaculovirinae, Polydnaviridae, Ichnovirus, Iridoviridae, Bracovirus, Parvoviridae, Flavivirdae, Tagviridae, Bunyaviridae, Rhabdoviridae, Reoviridae, Bimaviridae, Picornaviridae, Tetraviridae and Nadoviridae.