Patent Application
20120251450
The present invention relates to compositions comprising nanoparticles that preferentially associate with diseased cells, and methods relating thereto.
Nanoparticles are of interest to scientists and researchers because nanoparticles often behave differently than bulk materials of the same composition. Nanoparticles have long been studied for uses in biomedicine from tissue engineering to diagnostics to drug delivery. Of interest are nanoparticles that are selective towards diseased cells and that can perform multiple functions including diagnostics, imaging, and therapeutics. To achieve selectivity and multiple functionality in a single nanoparticle, known biologically active agents and imaging agents are attached to the surface of the nanoparticle. In some cases, the nanoparticle may only be a platform with all utility imparted on the nanoparticle through the attachment of known agents, i.e., a targeting ligand. Nanoparticles, i.e., without a targeting ligand, that themselves are selective toward diseased cells, imaging agents, and therapeutic agents would be of great value to the biomedical community.
The present invention relates to the compositions and methods of nanoparticles that preferentially associate with diseased cells.
In one embodiment, the present invention provides a nanoparticle composition comprising a first oxide of a first metal and a dopant that comprises an ion or an atom of a second metal.
In one embodiment, the present invention provides a method comprising providing a plurality of nanoparticles, providing a diseased cell and a healthy cell, contacting the diseased cell and the healthy cell with the nanoparticle, and allowing the nanoparticle to preferentially associate with the diseased cell. The nanoparticle comprises a first oxide of a first metal and a dopant that comprises an ion or an atom of a second metal.
In one embodiment, the present invention provides a kit comprising a nanoparticle composition comprising a first oxide of a first metal and a dopant that comprises an ion or an atom of a second metal, and a set of instructions for use.
The features and advantages of the present invention will be readily apparent to those skilled in the art upon a reading of the description of the preferred embodiments that follows.
The following figures are included to illustrate certain aspects of the present invention, and should not be viewed as exclusive embodiments. The subject matter disclosed is capable of considerable modification, alteration, and equivalents in form and function, as will occur to those skilled in the art and having the benefit of this disclosure.
The present invention relates to the compositions and methods of nanoparticles that preferentially associate with diseased cells.
Of the many advantages of the present invention, the present invention provides nanoparticle compositions that preferentially associate with diseased cells to image, track, probe, diagnose, and/or treat said diseased cells without the use of a targeting ligand. The term “preferentially associate” and its derivatives as used herein refers is a relative term meaning to associating with to a higher degree wherein associating may include external contact, internalization, or adsorption. Unless otherwise specified, preferential association compares diseased to healthy within the same environment. The preferential association can occur while imaging, tracking, probing, diagnosing, and/or treating diseased cells in vitro, in vivo, and/or ex vivo without the use of a targeting ligand. Known nanoparticle compositions similar to the nanoparticles of the present invention, e.g., Fe2O3, do not demonstrate said preferential association with diseased cells. Additionally, nanoparticles of the present invention may have multimodal imaging characteristics, dual imaging and diagnostic probing abilities, and enhanced cytotoxic effects. Together, with the preferential association with diseased cells, the nanoparticles of the present invention provide unique compositions and methods of use applicable to the biological sciences, environmental sciences, and chemical sciences.
In some embodiments, the present invention provides a nanoparticle composition comprising a first oxide of a first metal and a dopant that comprises an ion or an atom of a second metal.
In some embodiments, the present invention provides a method comprising providing a plurality of nanoparticles, providing a diseased cell and a healthy cell, contacting the diseased cell wand the healthy cell with the nanoparticle, and allowing the nanoparticle to preferentially associate with the diseased cell. The nanoparticle comprises a first oxide of a first metal and a dopant that comprises an ion or an atom of a second metal.
In some embodiments, the present invention provides a kit comprising a nanoparticle composition comprising a first oxide of a first metal and a dopant that comprises an ion or an atom of a second metal, and a set of instructions for use.
It should be understood that the term “nanoparticle” or “particle,” as used in this disclosure, includes all known shapes including, but not limited to, a sphere; a rod with a high to low aspect ratio; a wire; a star; a tetrapod or any other multi-legged shape; and a substantially spherical shape which may include an ovoid or a rice shape. Said shapes may be faceted.
In some embodiments, a nanoparticle of the present invention may comprise a metal oxide. In some embodiments, a nanoparticle of the present invention may consist essentially of a metal oxide. In some embodiments, a nanoparticle of the present invention may consist of a metal oxide.
In some embodiments, a nanoparticle may be a core-shell nanoparticle comprising a metal oxide core and at least one metal oxide layer. As used herein, “core-shell” as a descriptor of a nanoparticle shall mean a core nanoparticle with at least one shell encapsulating or substantially encapsulating a core nanoparticle. Core-shell nanoparticles may have multiple shell layers, which may be described or referred to as “onion” or “onionated” core-shell nanoparticles. In some embodiments, the core and adjacent layers in a core-shell nanoparticle may be different materials, e.g., different oxides.
It should be understood that “nanoparticle” as used herein, unless otherwise specified, is a generic term encompassing a nanoparticle without a shell, a core-shell nanoparticle, and an onionated nanoparticle.
It should be noted that when “about” is provided at the beginning of a numerical list, “about” modifies each number of the numerical list.
Nanoparticles may have diameters ranging from a lower limit of about 0.5 nm, 1 nm, 5 nm, 10 nm, 25 nm, 50 nm, 100 nm, or 250 nm to an upper limit of about 500 nm, 400 nm, 300 nm, 250 nm, 100 nm, 50 nm, 25 nm, or 10 nm, wherein the diameter may range from any lower limit to any upper limit to the extent that the selected range encompasses any subset between the upper and lower limits.
In some embodiments of the present invention, a core-shell nanoparticle may comprise a metal oxide core and at least one metal oxide layer. Depending on the embodiment, the metal oxide of the core may be different from the oxide in the shell. The core metal oxide nanoparticle may have a diameter ranging from a lower limit of about 0.5 nm, 1 nm, 5 nm, 10 nm, 25 nm, 50 nm, 100 nm, or 250 nm to an upper limit of about 500 nm, 400 nm, 300 nm, 250 nm, 100 nm, 50 nm, 25 nm, or 10 nm, wherein the diameter may range from any lower limit to any upper limit to the extent that the selected range encompasses any subset between the upper and lower limits. Depending on the embodiment, the metal oxide layer may have a thickness ranging from a lower limit of about 0.5 nm, 1 nm, 5 nm, 10 nm, 25 nm, or 50 nm to an upper limit of about 100 nm, 50 nm, 25 nm, 10 nm, or 5 nm, wherein the thickness may range from any lower limit to any upper limit to the extent that the selected range encompasses any subset between the upper and lower limits. For onionated core-shell nanoparticles, individual layers may be different thicknesses and different oxide compositions.
Suitable metal oxide materials for a nanoparticle of the present invention, the core of a core-shell nanoparticle of the present invention, and the shell(s) of an onionated core-shell nanoparticle of the present invention may be any known metal oxide nanoparticle, including, but not limited to, an oxide of magnesium, calcium, strontium, barium, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, molybdenum, ruthenium, palladium, silver, cadmium, tungsten, neodymium, gadolinium, hofimium, erbium, aluminum, silicon, gallium, germanium, indium, tin, lead, all oxidation states thereof, and any combination thereof. In some embodiments, the core and subsequent shell(s) of a nanoparticle may be different oxides. One skilled in the art would understand that a metal oxide of the same metal in different oxidation states would be considered different oxides. By way of nonlimiting example, Fe2O3 compared to Fe3O4 and α-Fe2O3 compared to γ-Fe2O3 are considered different oxides. In some embodiments, a nanoparticle may be magnetic, paramagnetic, or superparamagnetic. In some embodiments a nanoparticle comprises hematite and/or magnetite in the context of this invention.
In some embodiments, a metal oxide core may comprise a fluorophore. Suitable fluorophores may be any known fluorophore including those suitable for use in biomedical applications. Representative fluorophores include, but are not limited to, a coumarin dye including hydroxycoumarin, methoxycoumarin, aminocoumarin; a member of the ALEXA® fluor family (a sulfonated organic dye, available from Sigma-Aldrich in St. Louis, Mo.); a cyanine dye including Cy3 and Cy5; a fluorescein dye including fluorescein isothiocyanate; a rhodamine dye including tetramethyltrhodmine-5-(and 6)-isothiocyanate; a magnesium dye; a metal ligand complex; derivatives thereof; and combinations thereof.
In some embodiments, a nanoparticle may be synthesized by any known means including forced hydrolysis, precipitation, condensation, ball milling, and combinations thereof. In some embodiments where a core-shell or onionated nanoparticle is desired, a one-pot synthesis may be possible by synthesizing a core nanoparticle first then in the same reaction vessel forming a shell or multiple shells on the core nanoparticle.
Suitable forced hydrolysis methods may use water to force hydrolysis of at least one transition metal salt. By way of nonlimiting example, force hydrolysis may be carried out by dissolving at least one transition metal salt in an excess of solvent, adding a volume of water, and raising the temperature of the solution. The ratio of water to transition metal salt may effect the size of the resultant nanoparticle. Suitable solvents for the forced hydrolysis may be any known solvent capable of dissolving transition metal salts and achieving a desired elevated temperature. Examples of such solvents include, but are not limited to, a glycol, an ether, an alcohol, and combinations thereof. Suitable transition metal salts may be a salt of any transition metal with any known counter anion wherein the counter anion may include, but not be limited to, a fluoride, a chloride, a bromide, an iodide, a perchlorate, a chlorate, a chlorite, a hyperchlorite, a nitride, a nitrate, a nitrite, a sulfide, a sulfate, a sulfite, an acetate, an acetylacetonate, a carbonate, a formate, a phosphate, a cyanate, a thiocyanate, derivatives thereof, and combinations thereof. Suitable temperatures for forced hydrolysis include temperatures ranging from a lower limit of about 120° C., 130° C., 140° C., 150° C., or 160° C. to an upper limit of about 200° C., 190° C., 180° C., 170° C., 160° C., or 150° C., wherein the temperature may range from any lower limit to any upper limit to the extent that the selected encompasses any subset between the upper and lower limits. In embodiments where the nanoparticle of the present invention is a core-shell nanoparticle, the solution of the transition metal salts may further include a suspended nanoparticle. Hydrolyzed transition metals may form a shell encapsulating or substantially encapsulating the provided nanoparticle to form a core-shell or an onionated nanoparticle.
Suitable precipitation methods may precipitate at least one transition metal salt by heating a solution of the transition metal salt dissolved in a solvent. Suitable temperature for precipitation include temperatures ranging from a lower limit of about 120° C., 130° C., 140° C., 150° C., or 160° C. to an upper limit of about 200° C., 190° C., 180° C., 170° C., 160° C., or 150° C., wherein the temperature may range from any lower limit to any upper limit to the extent that the selected encompasses any subset between the upper and lower limits. Suitable solvents for the precipitation may be any known solvent capable of dissolving transition metal salts and achieving a desired elevated temperature. Examples of such solvents include, but are not limited to, a glycol, an ether, an alcohol, and combinations thereof. Suitable transition metal salts may be a salt of any transition metal with any known counter anion wherein the counter anion may include, but not be limited to, a fluoride, a chloride, a bromide, an iodide, a perchlorate, a chlorate, a chlorite, a hyperchlorite, a nitride, a nitrate, a nitrite, a sulfide, a sulfate, a sulfite, an acetate, an acetylacetonate, a carbonate, a formate, a phosphate, a cyanate, a thiocyanate, derivatives thereof, and combinations thereof. In embodiments where the nanoparticle of the present invention is a core-shell nanoparticle, the solution of the transition metal salts may further include a suspended nanoparticle. Precipitated transition metals may form a shell encapsulating or substantially encapsulating the provided nanoparticle to form a core-shell or an onionated nanoparticle.
Suitable condensation reactions may include at least one transition metal salt and heat to form a nanoparticle. Suitable temperature for condensation include temperatures ranging from a lower limit of about 120° C., 130° C., 140° C., 150° C., or 160° C. to an upper limit of about 200° C., 190° C., 180° C., 170° C., 160° C., or 150° C., wherein the temperature may range from any lower limit to any upper limit to the extent that the selected encompasses any subset between the upper and lower limits. Suitable solvents for the condensation may be any known solvent capable of dissolving transition metal salts and achieving a desired elevated temperature. Examples of such solvents include, but are not limited to, a glycol, an ether, an alcohol, and combinations thereof. Suitable transition metal salts may be a salt of any transition metal with any known counter anion wherein the counter anion may include, but not be limited to, a fluoride, a chloride, a bromide, an iodide, a perchlorate, a chlorate, a chlorite, a hyperchlorite, a nitride, a nitrate, a nitrite, a sulfide, a sulfate, a sulfite, an acetate, an acetylacetonate, a carbonate, a formate, a phosphate, a cyanate, a thiocyanate, derivatives thereof, and combinations thereof. In embodiments where the nanoparticle of the present invention is a core-shell nanoparticle, the solution of the transition metal salts may further include a suspended nanoparticle. Condensed transition metals may form a shell encapsulating or substantially encapsulating the provided nanoparticle to form a core-shell or an onionated nanoparticle.
In some embodiments, a nanoparticle may be synthesized or modified by a method that effects the surface charge of the nanoparticle, the size of the nanoparticle, the nanoparticle composition with the addition of a dopant, the band gap or redox potential of the nanoparticle, the degree of aggregation of the nanoparticle in a solution and/or suspension, or combinations thereof. Conditions that may be adjusted during synthesis include, but are not limited to, a solvent, a reaction temperature, a reaction time, a reactant, an additional reactant for a desired dopant, addition of an additive like a surfactant, and pH. Without being bound by theory or mechanism, it is believed that these parameters of a nanoparticle of the present invention may allow for influence reactive oxygen species production, a cytotoxic response to the nanoparticle, and/or a preferential association of the nanoparticle with a diseased cell.
In some embodiments, the surface charge of a nanoparticle may be changed after synthesis of the nanoparticle or controlled during synthesis of the nanoparticle. Surface charge may be measured by zeta potential. Suitable methods for changing the effective charge of a nanoparticle after synthesis of the nanoparticle include, but are not limited to, coating the nanoparticle with a polymer, surfactant, or surfmer; covalently functionalizing the nanoparticle; adjusting the pH of the suspension; and combinations thereof. Suitable methods for controlling the surface charge of a nanoparticle during synthesis of the nanoparticle include, but are not limited to, changing the solvent; adjusting the ratios of reactants to produce a nanoparticle with more than one metal oxide; including a polymer, surfactant, or surfmur in the synthesis solution; including a capping agent in the synthesis solution; adjusting the pH of the synthesis solution; and combinations thereof.
In some embodiments, a nanoparticle of the present invention may comprise a metal oxide and a dopant. In some embodiments, a nanoparticle of the present invention may consist essentially of a metal oxide and a dopant. In some embodiments, a nanoparticle of the present invention may consist of a metal oxide and a dopant. A dopant may be found in a metal oxide whether it be the metal oxide of a nanoparticle, a core, or a shell. Suitable dopants may include, but are not limited to, transition metal ions and/or transition metal atoms that replace a metal atom/ion of the metal oxide. In some embodiments, a dopant may be distributed substantially homogeneously throughout the metal oxide, as clusters within the metal oxide, as clusters at the surface of the metal oxide, as ions or atoms at the surface of the metal oxide, and combinations thereof. Suitable dopant materials include ions or atoms of any known metal, including, but not limited to, magnesium, calcium, strontium, barium, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, molybdenum, ruthenium, palladium, silver, cadmium, tungsten, neodymium, gadolinium, hofimium, erbium, aluminum, silicon, gallium, germanium, indium, tin, lead, all oxidation states thereof, and any combination thereof. In some embodiments, the metal oxide and the dopant may be different. One skilled in the art would understand the same metal in different oxidation state or crystal structure would be considered different.
In some embodiments, the band gap of a nanoparticle may be controlled to provide a nanoparticle with a desired redox potential. Suitable means of controlling or changing the band gap of a nanoparticle include doping with a suitable atom/ion; controlling the nanoparticle size; controlling the shell thickness of a core-shell and/or onionated nanoparticle; adjusting the oxygen stochiometry of the metal oxide during synthesis of the nanoparticle; adjusting the concentration and/or compositions of a species adsorbed to the surface of a nanoparticle, e.g., O2; adjusting the relative concentrations of different metal oxides that comprise the nanoparticle; and combinations thereof.
In some embodiments, suspendability and/or degree of aggregation of a nanoparticle may be modified by coating or substantially coating the nanoparticle with a polymer, surfactant, and/or surfmer. A polymer, surfactant, or surfmer may be any known polymer, surfactant, or surfmer including an anionic, a cationic, a nonionic, a Zwitter ion, and an ampholytic polymer, surfactant, or surfmer. In some embodiments, a monomer or partially polymerized polymer may be used to coat a nanoparticle allowing for in situ polymerization to form a coating around the nanoparticle. In some embodiments, suspendability and/or degree of aggregation of a nanoparticle may be modified by covalently functionalizing the nanoparticle.
In some embodiments, a nanoparticle of the present invention may contain a fluorophore within at least a portion of the nanoparticle matrix. In some embodiments, a fluorophore may be incorporated into at least a portion of a nanoparticle thru covalent functionalization to a reactant that forms the core nanoparticle, being present in the synthesis solution to be noncovalently incorporated in the core nanoparticle, adsorption and/or intercalation onto and/or into the core nanoparticle after synthesis, and combinations thereof.
In some embodiments, a nanoparticle may be associated with a fluorophore external to the nanoparticle. In some embodiments, the association may be covalent or noncovalent. In some embodiments, the association may be thru covalent attachment to a nanoparticle surface, to a nanoparticle coating, to a group covalently functionalized to the nanoparticle surface, and combinations thereof. In some embodiments, the association may be noncovalently adsorbed to the nanoparticle surface, noncovalently adsorbed within a nanoparticle surface coating and/or a plurality of covalent functional groups on the nanoparticle surface, and combinations thereof. In some embodiments, a fluorophore external to the nanoparticle and a fluorophore associated with a core nanoparticle may be the same or different.
In some embodiments, to the extent that increased specificity is desired, a targeting ligand may be associated with a nanoparticle. In some embodiments, the targeting ligand may be associated to the nanoparticle through direct covalent functionalization, covalent functionalization with a linker molecule, covalent functionalization to a molecule or polymer noncovalently associated with the nanoparticle, noncovalently associate with the nanoparticle, and combinations thereof. As used herein, the term “targeting ligand” may include, but is not limited to, any molecule that has specificity to a marker expressed by a cancer cell or pathogen, either extracellularly (e.g., on the cell surface or secreted by the cell) or intracellularly. In certain embodiments, the targeting ligand is specific for a tumor antigen. In some embodiments, the targeting ligand may be specific for a pathogenic antigen. Examples of a suitable targeting ligand may include, but are not limited to, antibodies and fragments thereof, haptens, polypeptides, aptemers, oligonucleotides, anti-sense RNA, Peptide Nucleic Acids, proteins, chimeric and/or fusion proteins, and the like, and any combination thereof.
In some embodiments, the environment or microenvironment of the nanoparticle may be probed by qualitatively or quantitatively analyzing fluorescence from the nanoparticle whether the fluorescence be from a fluorophore, because of the size and/or structure of a nanoparticle, because of the size and/or structure of a core-shell nanoparticle, or a combination of both. Suitable environmental conditions to probe may include, but are not limited to, the pH; the temperature; the pressure; the presence or absence of a small molecule, an ion, a biomolecule, a macromolecule, an element, a chemical, and/or a pathogen; the concentration of a small molecule, an ion, a biomolecule, a macromolecule, an element, a chemical, and/or a pathogen; and/or the like. In some embodiments, the nanoparticle may probe the progression of a chemical reaction or a biological process. A nanoparticle may be used to probe such conditions in gas, liquid, or solid environments.
In some embodiments, fluorescence may be observed and/or measured by eye or with the assistance of a laser, a light, a microscope, a fluorometer, a camera, and combinations thereof. One skilled in the art would recognize the available devices and methods for observing and/or measuring fluorescence.
One skilled in the art would understand that a variety of nanoparticle concentrations may be used depending on the application and method of administration. By way of nonlimiting example, suitable nanoparticle concentrations may include concentrations ranging from a lower limit of about 2 nM, 10 nM, 100 nM, 250 nM, 1 μM, 10 μM, 100 μM, 250 μM, 1 mM, 10 mM, or 100 mM, to an upper limit of about 5M, 1 M, 250 mM, 100 mM, 10 mM, 1 mM, 250 μM, 100 μM, 10 μM, or 1 μM wherein the nanoparticle concentration may range from any lower limit to any upper limit to the extent that the selected encompasses any subset between the upper and lower limits.
In some embodiments, a nanoparticle of the present invention may be used in antibacterial applications.
As used herein, the term “bacteria” or “bacteria cell” refers to a single-celled, prokaryote microorganism. Examples of bacteria include, but are not limited to, Acetobacter aurantius, Acinetobacter baumannii, Actinomyces israelii, Agrobacterium radiobacter, Agrobacterium tumefaciens, Azorhizobium caulinodans, Azotobacter vinelandii, Anaplasma, Bacillus, Bacteroides, Bartonella, Bordetella, Borrelia burgdorferi, Brucella, Burkholderia, Calymmatobacterium granulomatis, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Ehrlichia chaffeensis, Enterobacter cloacae, Enterococcus, Escherichia coli, Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Haemophilus, Helicobacter pylori, Klebsiella pneumoniae, Lactobacillus, Legionella pneumophila, Listeria monocytogenes, Methanobacterium extroquens, Microbacterium multiforme, Micrococcus luteus, Moraxella catarrhalis, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Peptostreptococcus, Porphyromonas gingivalis, Pseudomonas aeruginosa, Rhizobium radiobacter, Rickettsia, Rochalimaea, Rothia dentocariosa, Salmonella, Shigella dysenteriae, Staphylococcus, Stenotrophomonas maltophilia, Streptococcus, Treponema, Vibrio, Wolbachia, Yersiniam, and species thereof.
In some embodiments, bacteria cells may be exposed to a nanoparticle of the present invention. Exposing a bacteria cell to a nanoparticle may result in the nanoparticle contacting the bacteria cell; the nanoparticle becoming associated with the bacteria cell; the nanoparticle becoming internalized within the bacteria cell; the nanoparticle becoming associated with the outside of the bacteria cell; death of the bacteria cell; changes in metabolic rate of the bacteria cell; changes in reproductive rate of the bacteria cell; and combinations thereof. In some embodiments, in order to expose a bacteria cell to a nanoparticle, the nanoparticle may be incorporated into a medium including, but not limited to, a composite, a fiber, a paint, a coating, a solution, a suspension, a gel, a spray, and combinations thereof.
One skilled in the art would understand that a variety of nanoparticle concentrations may be used depending on the application and method of administration. One skilled in the art would understand that dose concentration is dependent on the administration concentration. As used herein, dose concentration refers to the concentration of nanoparticles that a bacteria, a cell, a tissue, or the like is exposed to. As used herein administration concentration refers to the concentration of nanoparticles given to a bacteria, a cell, a tissue, a patient, or the like. By way of nonlimiting example, a patient may be administered a high concentration of nanoparticles yielding a much lower concentration dose concentration that a diseased cell in a tissue would be exposed to. Both the dose concentration and administration concentration may be varied based on a variety of factors including the patient, the illness being treated, and other factors that would make the treatment applicable to that patient and disease combination. Suitable nanoparticle administration concentrations may include concentrations ranging from a lower limit of about 2 nM, 10 nM, 100 nM, 250 nM, 1 μM, 10 μM, 100 μM, 250 μM, 1 mM, 10 mM, or 100 mM, to an upper limit of about 5M, 1 M, 250 mM, 100 mM, 10 mM, 1 mM, 250 μM, 100 μM, 10 μM, or 1 μM wherein the nanoparticle concentration may range from any lower limit to any upper limit to the extent that the selected encompasses any subset between the upper and lower limits. Suitable nanoparticle dose concentrations may include concentrations ranging from a lower limit of about 2 nM, 10 nM, 100 nM, 250 nM, 1 μM, 10 μM, 100 μM, 250 μM, 1 mM, 10 mM, or 100 mM, to an upper limit of about 250 mM, 100 mM, 10 mM, 1 mM, 250 μM, 100 μM, 10 μM, or 1 μM wherein the nanoparticle concentration may range from any lower limit to any upper limit to the extent that the selected encompasses any subset between the upper and lower limits.
In some embodiments, a nanoparticle may be used in conjunction with a diseased cell, e.g., for imaging, tracking, probing, diagnosing, and/or treating.
As used herein, the term “diseased cell” refers to a cell in an abnormal condition that affects the body of an organism. Diseased cells may be associated with any known disease including, but not limited to, cancer, autoimmune disease, infectious disease, and parasitic disease. Diseased cells may be an activated T cell whose healthy counterpart would be an unactivated, nonactivated, or resting, T cell.
In some embodiments, a nanoparticle may be used in conjunction with a cancer cell, e.g., for imaging, tracking, probing, diagnosing, and/or treating. As used herein, the terms “cancer” or “cancer cell” refers to a cell or cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth which generally forms a distinct mass that shows partial or total lack of structural organization and functional coordination with normal tissue. The terms are meant to encompass benign growth (i.e., nonmalignant or normeoplastic growths), hematopoietic neoplasms (e.g., lymphomas or leukemias) as well as solid neoplasms (e.g., sarcomas or carcinomas), including all types of pre-cancerous and cancerous growths, or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. Hematopoietic neoplasms are malignant tumors affecting hematopoietic structures (structures pertaining to the formation of blood cells) and components of the immune system, including leukemias (related to leukocytes (white blood cells) and their precursors in the blood and bone marrow) arising from myeloid, lymphoid or erythroid lineages, and lymphomas (relates to lymphocytes). Solid neoplasms include sarcomas, which are malignant neoplasms that originate from connective tissues such as muscle, cartilage, blood vessels, fibrous tissue, fat or bone. Solid neoplasms also include carcinomas, which are malignant neoplasms arising from epithelial structures (including external epithelia (e.g., skin and linings of the gastrointestinal tract, lungs, and cervix), and internal epithelia that line various glands (e.g., breast, pancreas, thyroid). Examples of neoplasms that are particularly susceptible to treatment by the methods of the disclosure include leukemia, and hepatocellular cancers, sarcoma, vascular endothelial cancers, breast cancers, central nervous system cancers (e.g., astrocytoma, gliosarcoma, neuroblastoma, oligodendroglioma and glioblastoma), prostate cancers, lung and bronchus cancers, larynx cancers, esophagus cancers, colon cancers, colorectal cancers, gastro-intestinal cancers, melanomas, ovarian and endometrial cancer, renal and bladder cancer, liver cancer, endocrine cancer (e.g., thyroid), and pancreatic cancer. A cancer or tumor is treated or diagnosed according to the present methods. “Cancer” or “tumor” is intended to include any neoplastic growth in a patient, including an initial tumor and any metastases. The cancer can be of the liquid or solid tumor type. Liquid tumors include tumors of hematological origin, including, e.g., myelomas (e.g., multiple myeloma), leukemias (e.g., Waldenstrom's syndrome, chronic lymphocytic leukemia, other leukemias), and lymphomas (e.g., B-cell lymphomas, non-Hodgkins lymphoma). Solid tumors can originate in organs, and include cancers such as lung, breast, prostate, ovary, colon, kidney, and liver. As used herein, cancer cells, including tumor cells, refer to cells that divide at an abnormal (increased) rate. Cancer cells include, but are not limited to, carcinomas, such as squamous cell carcinoma, basal cell carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, undifferentiated carcinoma, bronchogenic carcinoma, melanoma, renal cell carcinoma, hepatoma-liver cell carcinoma, bile duct carcinoma, cholangiocarcinoma, papillary carcinoma, transitional cell carcinoma, choriocarcinoma, semonoma, embryonal carcinoma, mammary carcinomas, gastrointestinal carcinoma, colonic carcinomas, bladder carcinoma, prostate carcinoma, and squamous cell carcinoma of the neck and head region; sarcomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, synoviosarcoma and mesotheliosarcoma; hematologic cancers, such as myelomas, leukemias (e.g., acute myelogenous leukemia, chronic lymphocytic leukemia, granulocytic leukemia, monocytic leukemia, lymphocytic leukemia), and lymphomas (e.g., follicular lymphoma, mantle cell lymphoma, diffuse large Bcell lymphoma, malignant lymphoma, plasmocytoma, reticulum cell sarcoma, or Hodgkins disease); and tumors of the nervous system including glioma, meningoma, medulloblastoma, schwannoma or epidymoma.
In some embodiments, a nanoparticle may be used in conjunction with an autoimmune cell, e.g., for imaging, tracking, probing, diagnosing, and/or treating. As used herein, the term “autoimmune diseased cells” or “autoimmune cells,” includes cells that are defective in protection from apoptosis. This defect in protection from apoptosis may be in the pathway linked to TNF-induced apoptosis, or an apoptotic pathway unrelated to TNF. Autoimmune cells of the present invention include, but are not limited to, adult splenocytes, T lymphocytes, B lymphocytes, and cells of bone marrow origin, such as defective antigen presenting cells of a mammal. Autoimmune cells may be those associated with an autoimmune disease including, but are not limited to, psoriasis, rheumatoid arthritis, Crohns disease, and Lupus.
As used herein, the term “subject” and “patient” are used interchangeably herein and refer to both human and nonhuman animals and insects. The term “nonhuman animals” as used herein includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chicken, amphibian, fish, reptile, and the like. The term “insects” as used herein includes all arthropods that have a chitinous exoskeleton, a three-part body (head, thorax, and abdomen), three pairs of jointed legs, compound eyes, and two antennae, e.g., bees, flies, Drosophila flies, beetles, and the like.
Nanoparticles of the present invention may preferentially associate with diseased cells over healthy cells without the assistance of a targeting ligand. Without being bound by theory or mechanism, it is believed that the preferential association and/or preferential killing of diseased cells over healthy cells stems from the characteristics of the nanoparticle of the present invention. Examples of such characteristics may include the surface charge of the nanoparticle, the size of the nanoparticle, the nanoparticle composition with the addition of a dopant, the band gap or redox potential of the nanoparticle, the degree of aggregation of the nanoparticle in a solution and/or suspension, or combinations thereof. These factors may also lead to enhanced cytotoxic effects in addition to preferential association with diseased cells.
In some embodiments, a nanoparticle may associate preferentially with a diseased cell in vitro, in vivo, or ex vivo. In some embodiments, a nanoparticle may be associated with a diseased cell, and then transferred to an in vivo and/or ex vivo environment for an application or further analysis. For example, a nanoparticle of the present invention may be introduced to a diseased cell in vitro, then said diseased cell may be introduced in vivo, and finally said diseased cell may be removed for ex vivo analysis. Another example may be where a nanoparticle is introduced in vivo and allowed to associate with a diseased cell then the diseased cell is analyzed ex vivo.
In some embodiments, a nanoparticle of the present invention that is associated with a cell, diseased or healthy, may allow for detecting, imaging, diagnosing, and/or treating the cell. Suitable applications and methods that exploit these possibilities include determining the location of the cell, tracking the cell location, imaging processes within the cell, tracking intracellular and extracellular processes associated with the cell, diagnosing a disease, diagnosing a diseased state, treating a diseased cell, killing a diseased cell, inducing apoptosis in a diseased cell, increasing intracellular reactive oxygen species within the cell, inducing a cytotoxic effect to the cell, and combinations thereof.
In some embodiments, the location of a nanoparticle may be detected by imaging techniques including fluorescence, magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), and combinations thereof. In some embodiments, the detection of the location of a nanoparticle may enable applications including, but not limited to, nanoparticle tracking, cellular imaging, cellular tracking, ex vivo pathology, in vitro uptake, and in vivo pharmacodynamics. In some embodiments, a nanoparticle may have more than one fluorescent signal. In some embodiments, an imaging signal associated with a nanoparticle may interact with the environment or a chemical in the environment while a second imaging signal may not thereby allowing both tracking of location and/or measuring of concentration while monitoring a process or a condition of the environment of the nanoparticle. Such multimodal fluorescence may enable scientists or clinicians to study in vivo pharmacodynamics and pharmacokinetics in concert, to investigate intracellular processes, to diagnose diseased versus benign cells and their location in concert, to monitor the location and progress of cell death, and any other application where location and environment data may provide enhanced imaging, diagnostics, and/or therapeutics. Additional multi-modal fluorescence applications may be found in environmental science, environmental engineering, chemical engineering, and chemical physics.
In some embodiments, a nanoparticle of the present invention may be magnetic including but not limited to magnetic, paramagnetic, superparamagnetic, and combinations thereof. Magnetic fields may be used to control the location of the magnetic nanoparticle; to aggregate the magnetic nanoparticle; to direct the magnetic nanoparticle to a specific or general location; to detect the location of the magnetic nanoparticle; and combinations thereof. Magnetic nanoparticles associated with a cell may allow for using magnetic fields to detect the location of the cell; to move the cell; to manipulate the cell; to direct the movement of the cell; to direct the placement of the cell; to extract the cell from a given liquid media, cluster of cells, or tissue; to extract magnetic nanoparticle from the cell; and combinations thereof. In some embodiments, a nanoparticle may have magnetic properties and fluorescent properties that can be used in concert for any of the methods or any combination of methods disclosed herein.
One skilled in the art would understand that a variety of nanoparticle concentrations may be used depending on the application and method of administration. One skilled in the art would understand that dose concentration is dependent on the administration concentration. As used herein, dose concentration refers to the concentration of nanoparticles a bacteria, a cell, a tissue, or the like is exposed to. As used herein administration concentration refers to the concentration of nanoparticles given to a bacteria, a cell, a tissue, a patient, or the like. By way of nonlimiting example, a patient may be administered a high concentration of nanoparticles yielding a much lower dose concentration that a diseased cell in a tissue would be exposed to. Both the dose concentration and administration concentration may be varied based on a variety of factors including the patient, the illness being treated, and other factors that would make the treatment applicable to that patient and disease combination. Suitable nanoparticle administration concentrations may include concentrations ranging from a lower limit of about 2 nM, 10 nM, 100 nM, 250 nM, 1 μM, 10 μM, 100 μM, 250 μM, 1 mM, 10 mM, or 100 mM, to an upper limit of about 5M, 1 M, 250 mM, 100 mM, 10 mM, 1 mM, 250 μM, 100 μM, 10 μM, or 1 μM wherein the nanoparticle concentration may range from any lower limit to any upper limit to the extent that the selected encompasses any subset between the upper and lower limits. Suitable nanoparticle dose concentrations may include concentrations ranging from a lower limit of about 2 nM, 10 nM, 100 nM, 250 nM, 1 μM, 10 μM, 100 μM, 250 μM, 1 mM, 10 mM, or 100 mM, to an upper limit of about 250 mM, 100 mM, 10 mM, 1 mM, 250 μM, 100 μM, 10 μM, or 1 μM wherein the nanoparticle concentration may range from any lower limit to any upper limit to the extent that the selected encompasses any subset between the upper and lower limits.
A “kit” is any article of manufacture (e.g., a package or container). In some embodiments, a kit may comprise a nanoparticle of the present invention and a set of instructions. A kit may include a nanoparticle or a plurality of nanoparticles. A nanoparticle may be provided in a dry form or a wet form. Suitable dry forms include a powder, a crystal, a composite comprising a nanoparticle, and combinations thereof. Suitable wet forms include a suspension, a slurry, a solution, a paste, and combinations thereof. A kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention.
A kit may be for employing a nanoparticle of the present invention without cells, in vitro, in vivo, and/or ex vivo. A nanoparticle may be administered to a patient, an animal, a bacteria, a cell, a tissue, and/or a plant. A kit may also include a means of administering the nanoparticle including, but not limited to, intravenous, intraperitoneal, intragastric, oral, intra-tumoral, topical, and combinations thereof. A kit may be used by a technician, a scientist, a student, a clinician, a nurse, and/or the like. One of ordinary skill in the art would understand additional components of a kit based on a desired imaging, analysis, tracking, diagnostic, and/or treatment application.
One skilled in the art would understand that a variety of nanoparticle concentrations may be used depending on the application and method of administration. One skilled in the art would understand that dose concentration is dependent on the administration concentration. Suitable nanoparticle administration concentrations may include concentrations ranging from a lower limit of about 2 nM, 10 nM, 100 nM, 250 nM, 1 μM, 10 μM, 100 μM, 250 μM, 1 mM, 10 mM, or 100 mM, to an upper limit of about 5M, 1 M, 250 mM, 100 mM, 10 mM, 1 mM, 250 μM, 100 μM, 10 μM, or 1 μM wherein the nanoparticle concentration may range from any lower limit to any upper limit to the extent that the selected encompasses any subset between the upper and lower limits. Suitable nanoparticle dose concentrations may include concentrations ranging from a lower limit of about 2 nM, 10 nM, 100 nM, 250 nM, 1 μM, 10 μM, 100 μM, 250 μM, 1 mM, 10 mM, or 100 mM, to an upper limit of about 250 mM, 100 mM, 10 mM, 1 mM, 250 μM, 100 μM, 10 μM, or 1 μM wherein the nanoparticle concentration may range from any lower limit to any upper limit to the extent that the selected encompasses any subset between the upper and lower limits.
In some embodiments, a nanoparticle may be in an undesired location and/or environment including, but not limited to, nature including a body of water, an aquifer, a stream, a soil, a clay, a plant, and the like; or a living organism including a human, a mammal, a vertebrate, a non-vertebrate, a bacteria, a virus, and the like. In some embodiments, a nanoparticle may produce or enhance the production of reactive oxygen species. To mitigate against and/or treat for a deleterious effect of a nanoparticle, the nanoparticle may be removed from the undesired location and/or environment. To mitigate against and/or treat for a deleterious effect of a nanoparticle, an agent may be allowed to interact with the nanoparticle or the environment effected by the nanoparticle. Suitable agents include an antioxidant, a reactive oxygen species scavenger, a base, an acid, a solvent, a polymer, a surfactant, and/or a surfmer.
To facilitate a better understanding of the present invention, the following examples of preferred embodiments are given. In no way should the following examples be read to limit, or to define, the scope of the invention.
ZnO nanoparticles were formed by two methods. “ZnO I” nanoparticles were synthesized by forced hydrolysis of zinc acetate in diethylene glycol while “ZnO II” nanoparticles were synthesized in a similar manner using denture ethanol rather than diethylene glycol as the reaction media. The zeta potential, which measures surface charge, of both particles were measured to be ZnO I=+25 to +50 mV and ZnO II=−5 to −5 mV. Jukat leukemia T cells were exposed to each of the nanoparticles and viability was assessed.
Effect of Doping with a Reactive Oxygen Species (ROS) Catalyst.
ZnO nanoparticles doped with varying amounts of iron were prepared by co-precipitation of a zinc acetate and an iron acetate. For comparison, ZnO nanoparticles were prepared by similar procedure. Both nanoparticles Fe/ZnO and ZnO had the same diameter of ˜6.8 nm and similar zeta potential of +30 to +38 mV for Fe/ZnO and +40 mV for ZnO. Jukat leukemia T cells were exposed to each of the nanoparticles where intracellular superoxide concentration was assessed via MITOSOX™ Red detection (a mitochondrial superoxide indicator, available from Invitrogen, Carlsbad, Calif.) and viability. Superoxide concentration tracks well with viability. Interestingly, 2.5% to 10% iron doped ZnO nanoparticles are more effective at producing ROS killing the cells than similar ZnO nanoparticles without Fe doping. This may indicated that when a dopant is introduced into a nanoparticle of the present invention, a range of percent doping may be most effective. Additionally, it may be the case that the most effective percent range depends on the nanoparticle compositions and the dopant composition.
Increasing Nanoparticle Dispersion.
ZnO nanoparticles of ˜8.5 nm diameter were synthesized in diethylene glycol at 180° C. After synthesis, poly(acrylic acid) was added to the nanoparticles creating at least a partial coating which reduces the charge of the ZnO nanoparticle to neutral or slightly negative. Spectrophotometry experiments indicate that the dispersion of the ZnO nanoparticles increase with increased amounts of poly(acrylic acid). The cytotoxic effect of ZnO nanoparticles with varying amounts of poly(acrylic acid) was tested in Jukat leukemia T cells as assayed by flow cytometry and propidium iodide staining.
Changing Multiple Parameters to Enhance Cytotoxicity.
A series of ZnO I and ZnO II nanoparticles from above were synthesized with varying concentrations of iron dopant. Each of the nanoparticles were tested for cytotoxic effects in Jukat leukemia T cells as assayed by flow cytometry and propidium iodide staining.
Synthesis of FITC/SiO2—ZnO Particles.
FITC/SiO2—ZnO particles were synthesized by forced hydrolysis and condensation of FITC-binding silane and silicate to obtain the FITC-SiO2 core, followed by the formation of ZnO surface layer using zinc salt. In a typical synthesis, 10 mg (0.026 mmol) of FITC was dissolved in 3.0 mL ethanol by stirring for 20 min, followed by the addition of 20 μL (0.085 mmol) of vacuum-distilled (3-aminopropyl)-trimethoxysilane (APTMS). The reaction continued for 24 h under stirring in the dark. The amino group of the APTMS reacts with the isothiocyanate group of FITC to form N-1-(3-triethoxysilylpropyl)-N′-fluoresceylthiourea, and the resulting solution is referred to as FITC-APTMS. Second, 0.77 g tri-n-octylphosphine oxide (TOPO), 1.0 g polyethylene glycol (PEG), and 0.5 mL of FITC-APTMS were added to a flask containing 200 mL of diethylene glycol (DEG) solution and stirred for 10 min. Then, 0.5 mL of tetraethylorthosilicate (TEOS), 2.0 mL of water, and 1.5 mL of ammonium hydroxide (28-30%) were added into the above mixture and stirred for 1:5 h to form FITC/SiO2 cores. A part of this sample was separated at this point to obtain FITC encapsulated silica particles to compare their properties with FITC/SiO2 encapsulated ZnO particles. The resulting mixture was then heated to 100° C. and 2.6 g of zinc acetylacetonate, Zn(CH3COCHCOCH3)2 was introduced. Following this, the mixture was heated to 160° C. and maintained at that temperature for 2 h. The heating was then stopped to allow the mixture to cool down to room temperature with continuous stirring for 1 h. The resulting FITC/SiO2—ZnO particles were purified by centrifugation at 10,000 rpm for 8 min. The supernatant was removed and replaced with ethanol. This process was repeated for several times until no yellow fluorescence of FITC was observed in the supernatant. Subsequently, the resultant particles were dried in an oven.
Characterization of Core-Shell Nanoparticles.
The morphology, size, structure, and composition of FITC/SiO2—ZnO particles were thoroughly investigated using transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and UV-vis spectroscopy. In addition to the as-prepared samples, XPS spectra were also collected after removing 2, 5, 8, and 11 nm thick layers successively via Ar+ ion sputtering using a 2 kV Ar+ ion beam rastered over a 4 mm diameter sample area during 2 RPM sample rotation.
The FITC encapsulated fluorescent ZnO particles were light orange in color.
The inset of
X-ray photoelectron spectroscopy (XPS) measurements were carried out to investigate the core-shell architecture of the fluorescent ZnO particles through layer-by-layer sputtering.
Recalling that FITC is bound covalently to the silica matrix via the (3-aminopropyl)-trimethoxysilane (APTMS) coupling agent, the results shown in
The binding energy shifts of the Zn 2p3/2 peak and the Si 2p peak in the 11 nm sputter removed sample are not due to any random changes or charging effects because (i) the sample charging has been continuously compensated during the sputtering process by comparing the effect on known standards, (ii) if the binding energy shifts are due to any charging effect, it should display a gradual and systematic shift as sputtering (or charging) progresses, (iii) the peak shifts at 11 nm sputtering is associated with large changes in the intensities also indicating that the effect is related to transition between layers and the associated changes in the concentration of the elemental constituents, and (iv) the change in the XPS peaks at 11 nm sputtering coincides with the appearance of the N 1s signal from the FITC layer, again suggesting that the observed binding energy changes are associated with changes in the chemical environment/binding as data is collected progressively from different layers.
The optical properties of FITC/SiO2—ZnO particles in water were studied using UV-vis-NIR spectrophotometry in the absorption mode. The spectrum of the FITC/SiO2—ZnO samples was the sum of the spectral features observed in pure samples of ˜10 nm ZnO particles, ˜150 nm SiO2, and pure FITC measured separately, as illustrated in the absorption spectra in
The features near 362 nm in pure ZnO and FITC/SiO2—ZnO are the well expected absorption edges corresponding to the band gap of ZnO. The absorption peak at 489 nm of the encapsulated FITC molecules was lower than 494 nm observed for pure FITC. This small blue shift could be the result of covalent binding of the FITC molecules on the silica shell as also evident from the XPS data discussed earlier, and/or the presence of electron rich ZnO surface layer that might electromagnetically interact with the fluorophore. The charges in ZnO and/or near the polar ZnO—SiO2 interface might give rise to long-range electrostatic potential, which might extend through the silica layer to the FITC core. Another possible reason might be a direct binding of a fraction of the FITC molecules distributed in the silica shell (through the pores present in the silica layer) with ZnO nanocrystals. Additionally, UV-vis-NIR measurements were conducted for the FITC/SiO2—ZnO particles after treating with 1.5% NaOH to release the encapsulated dye. The NaOH addition dissolves the ZnO layer as evident from the disappearance of the band edge (
Fluorescent Emission Characteristics.
The fluorescence properties and stability of FITC/SiO2—ZnO particles were studied using fluorescence spectroscopy and flow cytometry. For flow cytometric analysis, a 3-color Epics XL cytometer (Coulter, Miami, Fla.) was used to evaluate the photobleaching and quenching effects of FITC/SiO2—ZnO particle aggregates. FITC/SiO2—ZnO particles were analyzed after keeping in oxygen-rich water for varying times, and in some cases, after subjecting the samples to illumination using a mercury lamp (Model SP200 spectrum tube, with 5000V and 10 mA output) and then resuspended in oxygen-rich water prior to analysis, and a minimum of 10,000 particle aggregates monitored for changes in relative fluorescence using a 488 nm argon laser.
Fluorescence spectra of FITC/SiO2—ZnO particles were measured at room temperature using a Horiba Jobin Yvon T64000 spectrometer equipped with Hamamatsu R943-02 GaAs photomultiplier. Two lines of a He—Cd laser, 441.6 and 325 run, were used for excitation. The photobleaching and quenching effects of the FITC/SiO2—ZnO nanoparticles were investigated by measuring the fluorescence data at fixed time intervals after subjecting the samples to low power laser light (wavelength 441.6 nm, power density ˜80 W cm-2). Similar data were also taken from pure FITC sample under identical conditions for comparison.
The FITC-ZnO particles are capable of emitting strong fluorescence both in the visible and UV wavelength ranges originating from FITC and ZnO layers, respectively.
Specifically,
Photostability.
The fluorescence emission of a fluorophore may be affected by the molecular interaction between the dye and various active species in the solvent such as dissolved oxygen. To investigate the environmental and photo-stability, the FITC/SiO2—ZnO particles were dispersed in oxygen-rich water and its fluorescence emission was recorded using a fluorescence spectrometer as a function of the time of laser exposure, shown in
Flow cytometry is a commonly used biological/biomedical research tool and the ability of the fluorescent FITC/SiO2—ZnO particles for use with this technique was carefully investigated (
A high percentage (98%) of the freshly synthesized FITC/SiO2—ZnO particle aggregates dispersed in oxygen-free water was derivatively fluorescent. As shown in
Utility Tests for Cell Imaging.
The feasibility of using FITC/SiO2—ZnO particles for particle tracking/cell imaging in biological environments was investigated using fluorescence confocal image microscopy (FCIM). Stationary phase E. coli cells were exposed to FITC/SiO2—ZnO particles and PI for 15 minutes at room temperature. FITC/SiO2—ZnO and PI exposed cells were spotted on a glass slide, allowed to air dry, and viewed using a Zeiss LSM 5 Pascal confocal microscope. Additional slides were prepared with E. coli cells exposed to either the FITC/SiO2—ZnO particles or propidium iodide (PI) alone to determine if there was any overlap in the fluorescence emission of the two dyes. The confocal microscope was configured to prevent detection of FITC fluorescence in the PI channel and vice versa. For cellular uptake and internalization studies, log phase Jurkat cells were adhered to poly-d-lysine treated glass bottom chamber slides (MatTek, Ashland, Mass.), treated with 0.25 mM FITC/SiO2—ZnO particles for 8 hours, washed three times in PBS/3% fetal bovine serum (FBS) to remove extracellular NP, stained with a PE-conjugated antibody specific to the CD3 cell surface protein (Beckman Coulter, Miami, Fla.) as previously described using 8 μl/200 μl of cells, and washed a final time in PBS/3% FBS. For confocal analysis, control slides were prepared to verify the absence of spectral overlap between the two dyes after appropriate instrument set-up.
We have investigated the ability of FITC/SiO2—ZnO particles (at 7.5 1.1 μg/mL) dispersed in saline medium to image E. coli using the green fluorescence of the particles (
Such a bivariate analysis allows for the discrimination of intact cells (FITC only) and dead/non-viable cells (FITC and PI). Visualization of the FITC/SiO2—ZnO signal alone indicates that the FITC/SiO2—ZnO particles were associated with the bacterial cells and emitting very bright green fluorescence (
Core-shell nanoparticle uptake and internalization studies were performed on eukaryotic Jurkat T cells as an example system. The cells were treated with FITC/SiO2—ZnO particles (green fluorescence) for 8 h, and then washed extensively to remove unattached extracellular particles and reduce background staining. Cells were then stained with a PE-conjugated antibody directed against the CD3 membrane-bound protein (red fluorescence) and confocal images taken using live cells to avoid internalization artifacts resulting from cell fixation.
Antibacterial Capacity.
We have recently shown that ZnO nanoparticles can selectively kill certain bacteria including E. coli and S. aureus. FITC/SiO2—ZnO particles were resuspended in sterile 0.9% NaCl aqueous solution, then sonicated for 15 minutes in a bath sonicator and continuously agitated by pipetting prior to dispensing to LB media for toxicity testing. For inhibitory threshold determination resuspended FITC/SiO2—ZnO particles were added to Luria-Bertani (LB) agar to different final concentrations (0-1250 μg/mL), as described in our earlier work. Time dependent toxicity tests were performed as follows. Equal densities of overnight E. coli cultures (based on OD600nm values) were used to inoculate LB broth with and without FITC/SiO2—ZnO particles. Broth cultures were incubated with shaking as above, sampled repeatedly, and viable cell densities measured via CFU enumeration by plating on particle free LB media.
Here we demonstrate a similar ability of FITC/SiO2—ZnO particles to inhibit the growth of these two organisms.
Selectivity Toward Cancer.
To determine whether FITC encapsulated particles with nanoscale ZnO outer surface can retain the ability of differentially killing cancer cells, new experiments were conducted. Toxicity of the FITC/SiO2—ZnO particles toward human T lymphocytes and Jurkat cancer cells was determined as follows. First, peripheral blood mononuclear cells (PBMC) were obtained by Ficoll-Hypaque (Histopaque-1077, Sigma, St. Louis, Mo.) gradient centrifugation using heparinized blood samples from healthy volunteers. This cell mixture was washed 3 times with Hank's buffer (Sigma), and incubated at 1.0×106 cells/mL in RPMI-1640 (Sigma) containing 10% fetal bovine serum. CD4+ T cells were subsequently isolated using negative immunomagnetic selection per manufacturer's instructions using a cocktail of antibodies against CD45RO, CD8, CD19, CD14, CD16, CD56, CD8, and glycophorin A (StemCell Technologies, Vancouver, B.C.) with collection of unlabeled T cells (typically >96% CD4+ and >93% viable as assessed by flow cytometry). Purified CD4+ T cells, or the Jurkat T cell line (ATCC, Rockville, Md.) were cultured in RPMI/10% FCS at 5×105 cells/mL in 96-well microliter plates and treated with various concentrations of FITC/SiO2—ZnO particles resuspended in PBS. For the delivery of FITC/SiO2—ZnO particles to cell cultures, a stock solution was made and sonicated for 10 minutes. Then immediately prior to dispensing into each individual cell culture well, particles were vortexed and immediately dispensed. This process was repeated for each culture well, to reduce differential particle delivery due to sedimentation. After 24 h of culture, cells were stained with propidium iodide (PI; BD Biosciences, San Jose, Calif.) to monitor loss of membrane integrity as previously reported and 10 μL, of fluorescently labeled microspheres (Molecular Probes, Eugene, Oreg.) added to each sample to allow for the absolute determination of cell numbers. Flow cytometry was used to analyze a minimum of 10,000 T cells per sample to determine changes in PI staining and quantification of cell death.
Flow cytometry was used to determine the number of viable human cancerous T cells compared to normal primary T cells after exposure to FITC/SiO2—ZnO particles for 24 h.
Therefore, the present invention is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. The particular embodiments disclosed above are illustrative only, as the present invention may be modified and practiced in different but equivalent manners apparent to those skilled in the art having the benefit of the teachings herein. Furthermore, no limitations are intended to the details of construction or design herein shown, other than as described in the claims below. It is therefore evident that the particular illustrative embodiments disclosed above may be altered, combined, or modified and all such variations are considered within the scope and spirit of the present invention. While compositions and methods are described in terms of “comprising,” “containing,” or “including” various components or steps, the compositions and methods can also “consist essentially of” or “consist of” the various components and steps. All numbers and ranges disclosed above may vary by some amount. Whenever a numerical range with a lower limit and an upper limit is disclosed, any number and any included range falling within the range is specifically disclosed. In particular, every range of values (of the form, “from about a to about b,” or, equivalently, “from approximately a to b,” or, equivalently, “from approximately a-b”) disclosed herein is to be understood to set forth every number and range encompassed within the broader range of values. Also, the terms in the claims have their plain, ordinary meaning unless otherwise explicitly and clearly defined by the patentee. Moreover, the indefinite articles “a” or “an,” as used in the claims, are defined herein to mean one or more than one of the element that it introduces. If there is any conflict in the usages of a word or term in this specification and one or more patent or other documents that may be incorporated herein by reference, the definitions that are consistent with this specification should be adopted.
