Patent Application
20140099256
Early tumor detection and treatment selection is paramount to achieving therapeutic success and long-term survival rates. At its early stage, many cancers are localized and can be treated surgically. However, well-defined tumor margins are often difficult to visualize with current imaging techniques. Highly-specific, molecular-targeted probes are needed for the early detection of molecular differences between normal and tumor cells, such as cancer-specific alterations in receptor expression levels. When combined with high-resolution imaging techniques, specific molecular-targeted probes will greatly improve detection sensitivity, facilitating characterization, monitoring and treatment of cancer.
Inorganic nanoparticles (NPs) hold great potential for revolutionizing current diagnostic techniques. However, potential risks of inorganic NPs on human health remain a big challenge and require further understanding. In order to minimize the toxicity induced by the accumulation of NPs in reticuloendothelial system (RES) organs, significant efforts have been devoted to developing renal clearable nanomaterials by manipulating their sizes, shapes and surface chemistries. For instance, pioneering work on the renal clearable quantum dots (QDs) showed that the zwitterionic cysteine coated QDs with a hydrodynamic diameter (HD) of 5.5 nm could be rapidly cleared out through the urinary system within 4 h and less than ˜5% of the QDs were found in the liver. The origin of such efficient renal clearance was due to the fact that zwitterionic ligands can behave like poly(ethylene glycol) (PEG) ligands to minimize serum protein adsorption while maintaining the small HD of the QDs. Although the emergence of the new renal clearable inorganic NPs potentially further advances the translation of inorganic NPs into clinical practices, the library of renal clearable NPs is still limited. It is an objective of this invention to develop nanoprobes that possess diverse material properties suitable for different imaging techniques and also exhibit optimal in vivo pharmacokinetics.
An embodiment of the invention is directed to a composition comprising a luminescent noble metal nanoparticle, wherein the surface of the noble metal nanoparticle is coated with a ligand, and wherein the noble metal nanoparticle is about 2 nm to 5 nm in diameter and further wherein a portion of the noble metal is present as its radioactive isotope. In an embodiment of the invention, the radioactive isotope is present at a concentration of up to 2% w/w of the noble metal.
The claimed invention provides nanoparticle compositions comprising a luminescent nanoparticle, methods for preparing the nanoparticle compositions and methods of using the nanoparticle compositions. The compositions of the present invention comprise noble metal nanoparticles, which are capable of emitting in the near infrared region of the light spectrum. The claimed invention provides for compositions comprising a luminescent noble metal nanoparticle. In an embodiment, the noble metal nanoparticle comprises between 2 and 1000 noble metal atoms. In preferred embodiments, the noble metal is selected from the group consisting of gold, silver, and copper.
In certain embodiments of the compositions of the claimed invention, a portion of the noble metal is present as its radioactive isotope. In an embodiment of the invention, the radioactive isotope is present at a concentration of up to 2% w/w of the noble metal. Embodiments of the invention comprise the radioactive isotopes of noble metal. In certain embodiments the radioactive isotope is 198Au. In other embodiments, the radioactive isotope is 64Cu. The presence of the radioactive isotope in the metal nanoparticle aids in the rapid monitoring of the pharmacokinetics of the NIR emitting radioactive particles and also offers an opportunity for in vivo SPECT imaging by emitting gamma rays.
The properties of the nanoparticles enable excretion through the kidneys, as well as selective uptake and retention in tumors compared with normal tissues. This, along with the lack of in vivo toxicity, has resulted in a composition that is promising for translation to the clinic.
In certain embodiments of the claimed invention, the surface of the luminescent noble metal nanoparticle is modified with a ligand. In certain embodiments, the surface of the nanoparticle is coated with a ligand that prevents adsorption of serum proteins on the surface of the nanoparticle and prevents fouling of the nanoparticle. In certain embodiments of the invention, the surface ligand is a protein or peptide. In other embodiments of the invention, the ligand is a polymer such as poly(ethylene glycol) (PEG) or oligo(ethylene glycol) (OEG). In certain embodiments of the invention, the anti-fouling ligand is a zwitterionic material such as sulfobetaine methacrylate (SBMA), carboxybetaine methacrylate (CBMA), poly(carboxybetaine acrylamide) (polyCBAA) or a mixed charge material. In certain embodiments of the invention the ligand is glutathione.
In certain embodiments, the ligand is capable of binding to at least one cellular component. The cellular component may be associated with specific cell types or having elevated levels in specific cell types, such as cancer cells or cells specific to particular tissues and organs. Accordingly, the nanoparticle can target a specific cell type, and/or provides a targeted delivery for the treatment and diagnosis of a disease. As used herein, the term “ligand” refers to a molecule or entity that can be used to identify, detect, target, monitor, or modify a physical state or condition, such as a disease state or condition. For example, a ligand may be used to detect the presence or absence of a particular receptor, expression level of a particular receptor, or metabolic levels of a particular receptor. The ligand can be, for example, a peptide, a protein, a protein fragment, a peptide hormone, a sugar (i.e., lectins), a biopolymer, a synthetic polymer, an antigen, an antibody, an antibody fragment (e.g., Fab, nanobodies), an aptamer, a virus or viral component, a receptor, a hapten, an enzyme, a hormone, a chemical compound, a pathogen, a microorganism or a component thereof, a toxin, a surface modifier, such as a surfactant to alter the surface properties or histocompatability of the nanoparticle or of an analyte when a nanoparticle associates therewith, and combinations thereof.
The present invention further encompasses methods of using the luminescent nanoparticles in order to study a biological state. The invention provides for a method of monitoring a molecule of interest by contacting the luminescent noble metal nanoparticle with a sample containing the molecule of interest. In a preferred embodiment, the molecule of interest is present in a biological sample.
Unless otherwise noted, the terms used herein are to be understood according to conventional usage by those of ordinary skill in the relevant art. It is to be understood that as used in the specification and in the claims, “a” or “an” can mean one or more, depending upon the context in which it is used. Thus, for example, reference to “a cell” can mean that at least one cell can be utilized.
The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the Working Examples included herein. However, before the present compounds, compositions, and methods are disclosed and described, it is to be understood that this invention is not limited to specific noble metals, specific conditions, or specific methods, etc., as such may, of course, vary, and the numerous modifications and variations therein will be apparent to those skilled in the art. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.
As used herein, the term “noble metal” refers to the group of elements selected from the group consisting of gold, silver, and copper and the platinum group metals (PGM) platinum, palladium, osmium, iridium, ruthenium and rhodium. In certain preferred embodiments of the present invention, the noble metal is selected from the group consisting of gold, silver, and copper. In other preferred embodiments, the noble metal is silver. In other preferred embodiments, the noble metal is gold. In other preferred embodiments, the noble metal is copper.
As used herein, the term “nanoparticle” refers to an association of 2-1000 atoms of a metal. Nanoparticles may have diameters in the range of about 2 to about 5 nm. In other preferred embodiments, the nanoparticles comprise approximately 2-1000, approximately 2-500, approximately 2-250, approximately 2-100, approximately 2-25 atoms, or approximately 2-10 atoms.
Compositions of the invention are capable of emitting in the near infra-red range of 700 nm to 1100 nm. Compositions of the claimed invention are capable of being detected by Single-photon emission computed tomography (SPECT) and fluorescence imaging techniques. Therefore, the nanoprobes of the claimed invention can serve as dual-modality imaging probes.
The present invention further encompasses methods for the preparation of the noble metal nanoparticle having the characteristics as described herein. In one embodiment, the method of preparing a noble metal nanoparticle comprises the steps of: a) combining an aqueous solution comprising a noble metal, and an aqueous solvent to create a combined solution; b) adding a first ligand; c) mixing the combined solution to allow the formation of a noble metal nanoparticle; and d) adjusting the pH of the combined solution using acid or base. In certain embodiments of the invention, the aqueous solution of the noble metal contains an amount of a radioactive isotope of the noble metal. In an embodiment of the invention, the radioactive isotope is present in the nanoparticle at a concentration of up to 2% w/w of the noble metal.
In certain embodiments of these methods, a reducing agent is added to the combined solution to reduce the noble metal nanoparticle. Preferably the reducing agent is selected from the group comprising a chemical reducing agent, light, or a combination thereof. In certain embodiments of these methods, light can be used as a reducing agent to photoreduce the noble metal nanoparticles. In certain other embodiments of these methods, a chemical reducing agent can be used as a reducing agent. In one embodiment, light is used in combination with a reducing agent to photoreduce the noble metal nanoparticles.
Preferably, the aqueous solution comprising a noble metal ion used in the preparation of the compounds is selected from the group consisting of AgNO3, HAuCl4.nH2O, and CuCl2.nH2O. In one embodiment, the aqueous solution comprising a noble metal is AgNO3.
In another embodiment, the aqueous solution comprising a noble metal is HAuCl4.nH2O. In a further embodiment, the aqueous solution comprising a noble metal is CuCl2.nH2O.
In one embodiment, the aqueous solution comprising a noble metal is HAuCl4.nH2O, a reducing agent is added to the combined solution along with a ligand, the pH adjusted, and the combined solution is mixed for at least one hour to allow the formation of the gold nanoparticle. In another embodiment, the pH adjusted, combined solution is mixed for about 48 hours or longer (up to several months) to allow the formation of a luminescent gold nanoparticle. In another embodiment, noble metal nanoparticles are created through photoreduction through irradiation with visible or ultraviolet light to allow the formation of a gold, silver or copper nanoparticle.
In an embodiment of the invention, a therapeutic agent is attached to the nanoparticle. The therapeutic agent is selected from the group consisting of antibiotics, antimicrobials, antiproliferatives, antineoplastics, antioxidants, endothelial cell growth factors, thrombin inhibitors, immunosuppressants, anti-platelet aggregation agents, collagen synthesis inhibitors, therapeutic antibodies, nitric oxide donors, antisense oligonucleotides, wound healing agents, therapeutic gene transfer constructs, extracellular matrix components, vasodialators, thrombolytics, antimetabolites, growth factor agonists, antimitotics, statin, steroids, steroidal and nonsteroidal anti-inflammatory agents, angiotensin converting enzyme (ACE) inhibitors, free radical scavengers, PPAR-gamma agonists, small interfering RNA (siRNA), microRNA, and anti-cancer chemotherapeutic agents.
In an embodiment of the invention, the compositions of the invention are used to monitor the surface of cell membranes. In certain embodiments of the invention, the cell membranes that are targeted are tumor cell membranes.
After administration of the nanoparticle to a subject, the blood residence half-time of the nanoparticles may range from about 2 hours to about 25 hours, from about 3 hours to about 20 hours, from about 3 hours to about 15 hours, from about 4 hours to about 10 hours, or from about 5 hours to about 6 hours. Longer blood residence half-time means longer circulation, which allows more nanoparticles to accumulate at the target site in vivo. Blood residence half-time may be evaluated as follows. The nanoparticles are first administered to a subject (e.g., a mouse, a miniswine or a human). At various time points post administration, blood samples are taken to measure nanoparticle concentrations through suitable methods.
An embodiment of the claimed invention is directed to a method for detecting a component of a cell comprising the steps of: contacting the cell with a composition comprising a luminescent noble metal nanoparticle, wherein the surface of the noble metal nanoparticle is coated with a ligand, and wherein the noble metal nanoparticle is about 2 nm to 5 nm in diameter, wherein a portion of the noble metal is present as a radionuclide; and monitoring the binding of the nanoparticle to the cell or a cellular component by at least one imaging technique.
A further embodiment of the invention is directed to a method for targeting a tumor cell comprising administering the tumor cell an effective amount of a composition comprising a luminescent noble metal nanoparticle, wherein the surface of the noble metal nanoparticle is coated with a ligand, and wherein the noble metal nanoparticle is about 2 nm to 5 nm in diameter, wherein a portion of the noble metal is present as a radionuclide, wherein the ligand is capable of binding a tumor marker; and at least one therapeutic agent.
An embodiment of the claimed invention is directed to a metal nanoparticle that is renal clearable. In certain embodiments of the invention, the compositions demonstrate greater than 50% renal clearance within 48 hours of administration.
Glutathione-Coated Luminescent Gold Nanoparticles (G-AuNPs) were synthesized by a self-dissociation of Au(I)-glutathione polymers in an aqueous solution. In a typical synthesis, a fresh aqueous solution containing reduced L-glutathione (25 mM) was added into a HAuCl4 aqueous solution (25 mM) containing 198Au at a 1:1 molar ratio of gold to thiolated ligand. The solution was centrifuged at 21,000 g for 1 minute to remove the insoluble aggregates as well as large NPs. The supernatant was further purified by adding a small amount of ethanol into the aqueous solution (the ratio between water and ethanol is 2:1). Under such conditions, the luminescent gold NPs were precipitated out of the solution while the free GSH and gold ions remained in the solution. The precipitates were then re-suspended in aqueous solution (DI water, PBS or 10% (v/v) FBS-containing MEM (without phenol red). The final solution contained G-AuNPs with diameter of ˜2 nm. The pH of solution was adjusted by 1 M NaOH or 1 M HCl and then measured by a pH meter.
CuNPs were synthesized by mixing 1 mmol 64CuCl2 and 4 mmol glutathione (GSH) in 40 mL de-ionized (DI) water, followed by adding 5 M NaOH solution to tune the pH of the mixture to about 7, and adding extra DI water to fix the total volume to 50 mL, which was then equally divided into 10 samples in 20 mL vials, which were closed and stored at room temperature for about one week till the solution color unchanged, and the blue Cu(II)-GSSG complex was obtained. The synthesized 64Cu(II)-GSSG complex solution of 4.8 mL was precipitated by adding 3 times volume ethanol (21,000 g, 1 min). Then, the precipitates were dried by N2 purge and redissolved in 45 mL DI water, followed by adding fresh prepared 2 mL 4.8 M NaBH4 solution under stirring with the speed of 1150 rpm for 5-10 min. Then, the pH of the solution was tuned to 4-5 by adding 5 M HCl solution, and the solution was kept at R.T. for ˜30 min under stirring to form 64Cu radioactive luminescent nanoparticles.
The pharmacokinetic parameters of GS-[198Au]AuNPs were measured in normal Balb/c mice. The results showed that the GS-[198Au]AuNPs exhibited a two compartment profile of in vivo kinetics with a t½, of 5.0 min and a t½β of 12.7 hours (
More than 50% of GS-[198Au]AuNPs were cleared out of the body after 48 hours (
The accumulation of the NPs in heart, lung, muscle and fat also decreased with time, indicating that the GS-[198Au]AuNPs were only temporally distributed to these tissues due to the rapid adsorption process and can be efficiently washed away within the first 48 hours without causing an uptake increase in the liver and spleen. The origin of the resulted biodistribution and efficient renal clearance of the NPs were attributed to the small particle size and glutathione ligand, which enable the GS-[198Au]AuNPs highly stable in the physiological environment and resistant to serum protein adsorption. As a result, the hydrodynamic diameter of the NPs in the body was still below the size threshold for renal clearance and the NPs remained stealthy to RES organs.
Although “being small” is generally seen as an efficient approach to boost the clearance of biomaterials from the body, the avidity between materials and serum proteins also plays an important role in the excretion. Seemingly a dilemma, it is hard to know which factors in the renal excretion of GS-CuNPs and Cu(II)-GSSG complex are reliably important. As such, the renal clearance kinetics and biodistribution of GS-CuNPs and Cu(II)-GSSG complex were evaluated in BALB/c mice (
The detailed biodistribution studies of mice injected with GS-CuNPs and Cu(II)-GSSG complexes were consistent with their renal clearance profiles (
The successful synthesis of renal clearable GS-CuNPs provided an exciting opportunity to explore a potential biomedical application of CuNPs in PET imaging because 64Cu is a well known β+ (0.653 MeV, 17.8%) emitter for PET imaging with a half-time of 12.7 h. In addition, considering copper is a necessary trace element in human body with the total content of 100-150 mg and ˜18 mg in the liver the trace amount of renal clearable CuNPs can be combined with highly sensitive nuclear imaging techniques, potential side effect induced by copper metal in the liver can be further minimized.
Although the present invention has been described in connection with some embodiments, it is not intended to be limited to the specific form set forth herein. Rather, the scope of the present invention is limited only by the accompanying claims. Additionally, although a feature may appear to be described in connection with particular embodiments, one skilled in the art would recognize that various features of the described embodiments may be combined in accordance with the invention. In the claims, the term comprising does not exclude the presence of other elements or steps.
Furthermore, although individually listed, a plurality of means, elements or method steps may be implemented by e.g. a single unit or processor. Additionally, although individual features may be included in different claims, these may possibly be advantageously combined, and the inclusion in different claims does not imply that a combination of features is not feasible and/or advantageous. Also, the inclusion of a feature in one category of claims does not imply a limitation to this category but rather indicates that the feature is equally applicable to other claim categories as appropriate. Furthermore, the order of features in the claims do not imply any specific order in which the features must be worked and in particular the order of individual steps in a method claim does not imply that the steps must be performed in this order. Rather, the steps may be performed in any suitable order. In addition, singular references do not exclude a plurality. Thus references to “a”, “an”, “first”, “second” etc do not preclude a plurality.
This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No. 61/694,145 filed Aug. 28, 2012, which is incorporated herein by reference in its entirety as if fully set forth herein.
This invention was made with government support under Grant Nos. R21EB009853 and R21EB011762 awarded by the National Institutes of Health and Grant No. RP120588 awarded by Cancer Prevention and Research Institute of Texas. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61694145 | Aug 2012 | US |
