1. Technical Field
This document relates to methods and materials involved in nanoparticles (e.g., rare earth nanorods). For example, this document relates to materials and methods involved in neodymium, samarium, europium, gadolinium, and terbium nanoparticles.
2. Background Information
Nanotechnology is a rapidly expanding into biomedical research. Nanobiotechnology is opening new avenues in bioimaging, medical diagnostics, and disease therapy. Bio-imaging with inorganic fluorescent nanoparticle probes recently attracted widespread interest in biology and medicine.
This document provides methods and materials related to rare earth particles such as rare earth nanorods (e.g., inorganic lanthanide hydroxide nanorods). For example, this document provides neodymium hydroxide [NdIII(OH)3], samarium hydroxide [SmIII(OH)3], europium hydroxide [EuIII(OH)3], gadolinium hydroxide [GdIII(OH)3], and terbium hydroxide [TbIII(OH)3] nanorods. These nanorods can be prepared using a microwave technique that is simple, fast, clean, efficient, economical, non-toxic, and eco-friendly. The europium hydroxide nanorods provided herein can be fluorescent, can enter cells, and can retain their fluorescent properties once they have entered cells. In addition, the europium hydroxide nanorods provided herein can be used to visualize the internalization of drugs or biomolecules attached to the nanorods into cells for imaging, therapeutic, and/or diagnostic purposes. The europium hydroxide nanorods provided herein can be non-toxic, fluorescent, inorganic, Europium(III) hydroxide nanorods and can be used as pro-angiogenic agents in vivo.
The process of angiogenesis can play a role in embryogenesis, wound healing, and tumor genesis through the growth of new blood vessels from pre-existing vasculature. The europium hydroxide nanorods provided herein can be used to promote angiogenesis in tissues such as ischemic tissues. In some cases, europium hydroxide inorganic fluorescent nanorods can be used as a pro-angiogenic agent instead of or in combination with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (BFGF). The europium hydroxide nanorods provided herein can be non-toxic nanorods as observed by a cell proliferation assay, a cell cycle assay, and/or a CAM assay and can induce endothelial cell proliferation. The europium hydroxide nanorods provided herein can be used to treat heart or limb ischemic tissues in humans. Like EuIII(OH)3 nanorods, NdIII(OH)3, SmIII(OH)3, and TbIII(OH)3 nanorods are non-toxic, as observed by a cell proliferation assay.
In comparison to organic dyes (Fluorescein, Texas Red™, Lissamine Rhodamine B, Tetramethylrhodamine, etc.) and fluorescent proteins (Green fluorescent protein, GFP), the inorganic fluorescent europium hydroxide nanorods provided herein can have several unique optical and electronic properties such as size- and composition-tunable emission from visible to infrared wavelengths, a large stokes shift, a symmetric emission spectrum, simultaneous excitation of multiple fluorescent colors, very high levels of brightness and photostability.
In general, one aspect of this document features a method for making europium hydroxide nanorods. The method comprises, or consists essentially of, microwave heating a mixture of LnIII(NO3)3 (where Ln=Nd, Sm, Eu, Gd, or Tb) and aqueous ammonium hydroxide. The lanthanide hydroxide [LnIII(OH)3] nanorods can be between 10 and 500 nm in length. The diameter of the lanthanide hydroxide nanorods can be between 1 and 100 nm.
In another aspect, this document features lanthanide hydroxide nanorods having a length between 10 and 500 nm and a diameter between 1 and 100 nm, wherein the nanorods promote angiogenesis.
In another aspect, this document features a method of promoting angiogenesis, wherein the method comprises, or consists essentially of, contacting cells with europium hydroxide nanorods. The europium hydroxide nanorods can be between 10 and 500 nm in length. The diameter of the europium hydroxide nanorods can be between 1 and 100 nm.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
This document provides methods and materials related to rare earth particles such as rare earth nanorods. For example, this document provides rare earth (e.g., lanthanide) particles such as neodymium (Nd), samarium (Sm), europium (Eu), gadolinium (Gd), and terbium (Tb) hydroxide nanorods, methods and materials for making rare earth particles (e.g., neodymium, samarium, europium, gadolinium, and terbium hydroxide nanorods), and methods and materials for using rare earth particles (e.g., neodymium, samarium, europium, gadolinium, and terbium hydroxide nanorods) as an imaging agent and/or to promote angiogenesis.
Lanthanide (e.g., neodymium, samarium, europium, gadolinium, and terbium) hydroxide nanoparticles (e.g., nanorods) provided herein can have any dimensions. For example, the europium hydroxide nanorods provided herein can have a length between 50 nm and 500 nm (e.g., between 100 nm and 400 nm, between 150 nm and 350 nm, and between 200 nm and 300 nm), and can have a thickness between 10 nm and 100 nm (e.g., between 20 nm and 90 nm, between 25 nm and 75 nm, or between 30 nm and 50 nm). Any appropriate method can be used to make lanthanide hydroxide nanoparticles. For example, a microwave technique such as that described herein can be used to make lanthanide (e.g., neodymium, samarium, europium, gadolinium, and terbium) hydroxide nanorods.
In some cases, the lanthanide hydroxide nanoparticles provided herein can be combined with drugs or other therapeutic agents for delivery to a mammal (e.g., a human). For example, a drug can be covalently linked to an europium hydroxide nanoparticle (e.g., nanorod). In such cases, the europium hydroxide nanoparticles (e.g., nanorods) can be used to track the location and/or concentration of the drug within a mammal. Examples of therapeutic agents that can be combined with lanthanide hydroxide nanoparticles include, without limitation, polypeptides, antibodies, C225, gemcitabine, cisplatin, and organic drug molecules containing an active functional group.
Any appropriate method can be used to combine lanthanide hydroxide nanoparticles with therapeutic agents. For example, a therapeutic agent can be conjugated to a lanthanide hydroxide nanoparticle. Before conjugating a therapeutic agent (e.g., a drug molecule) with a lanthanide hydroxide nanoparticle (e.g., a europium hydroxide nanorod), the surface of the nanoparticle (e.g., nanorod) can be modified with an active functional group (e.g., an amino or mercapto group). For example, aminopropyl trimethoxy silane (APTMS) or mercapto-propyl trimethoxy silane (MPTMS) can be used to functionalize the surface of lanthanide hydroxide nanorods, as described elsewhere (Feng et al., Anal. Chem., 75:5282-5286 (2003)). In some cases, nanoparticles (e.g., nanorods) can be functionalized using a microwave technique such as that described herein. Surface modified lanthanide hydroxide nanoparticles can be combined with different therapeutic agents (e.g., organic drug molecules, polypeptides, or antibodies) by covalent bond formation.
As described herein, lanthanide hydroxide nanoparticles such as europium hydroxide nanorods can be used to promote angiogenesis within a mammal. For example, a mammal can be identified as needing a pro-angiogenic agent. Once identified, lanthanide hydroxide nanoparticles provided herein can be administered to the mammal. Such an administration can be a systemic or local administration. For example, europium hydroxide nanorods can be directly injected into tissue in need of angiogenesis. Following administration, the mammal can be monitored to determine whether or not angiogenesis was promoted or to determine whether or not additional administrations are needed.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Neodium (III) nitrate hexahydrate (99.9%), Samarium (III) nitrate hexahydrate (99.99%), Europium (III) nitrate hydrate [Eu(NO3)3.xH2O, 99.99%], Gadolinium (III) nitrate hexahydrate (99.999%), Terbium (III) nitrate hexahydrate (99.999%), and aqueous ammonium hydroxide [aq.NH4OH, 28-30%] were purchased from Aldrich (USA) and were used without further purification. [3H] Thymidine was purchased from Amersham Biosciences (Piscataway, N.J.). Phosphate Buffered Saline (PBS) without calcium and magnesium was purchased from Cellgro Mediatech, Inc. (Herndon, Va.). Endothelial Cell Basal Medium (EBM), without anti-microbial agents, Trypsin/EDTA (0.25 mg/mL), Trypsin Neutralizing Solution (TNS), and a set of 5% of fetal bovine serum (FBS), 0.4% of bovine brain extract, and 0.1% of gentamicin sulfate amphotericin-B, were obtained from Cambrex Bio Science Inc. (Walkersville, Md.) and used to make EBM complete media. Falcon tissue culture dishes were purchased from Beckon Dickinson Labware (Beckon Dickinson and Company, N.J., USA). An in situ cell death detection kit, TMR red, for use in a Tunnel assay was purchased from Roche (Cat. No. #12 156 792 910). Monoclonal mouse IgG (Cat. No # OP72-100UG), anti-phospho map kinase (rabbit polyclonal IgG, Cat. No. # 07-467) antibody, and anti-mouse IgG or anti-rabbit IgG-HRP (Cat. # Sc-2301) were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif. USA). The Image-iT™ LIVE Green Reactive Oxygen Species (ROS) Detection Kit (I36007) was purchased from Invitrogen Molecular Probes (Eugene, Oreg.).
Europium Hydroxide [Eu(OH)3)] Nanorods
Lanthanide nitrate and aqueous ammonium hydroxide (28-30% A.C.S reagent) were purchased from Aldrich Co. and Sigma-Aldrich, respectively, and used as received without further purification. LnIII(OH)3, where Ln=Nd, Sm, Eu, Gd, or Tb, nanorods were prepared by microwave heating a mixture of an aqueous solution of Ln(III) nitrate and aq.NH4OH at atmospheric pressure in an open reflux system. In a typical synthesis, 10 mL of aqueous NH4OH was added to 20 mL 0.05 M of an aqueous solution of Ln(III) nitrate (pH=5.5) in a 100 mL round-bottomed flask. A colloidal precipitate, without any special morphology, was obtained upon the addition of NH4OH to Ln(III) nitrate solution. The pH of the solution before and after the reaction was 9.4 and 7.5, respectively. The samples were irradiated for 1 to 60 minutes with 60% of the instrument's power (on/off irradiation cycles ratio of 3/2) in order to control the reaction and reduce the risk of superheating of the solvent. The microwave refluxing apparatus was a modified domestic microwave oven (GOLD STARR 1000 W, LA Electronics, Inc., Huntsville, Ala.) with a 2.45 GHz output power, as described elsewhere (Matsumura Inoue et al., Chem. Lett., 2443 (1994)). In the post-reaction treatment, the resulting products were collected, centrifuged at 15000 rpm, washed several times using distilled water, and then dried overnight under vacuum at room temperature. The yield of the as-prepared products was more than 95% for all of the lanthanide hydroxide nanoparticles. The above experiments were conducted several times and exhibited good reproducibility.
The following cell culture experiments were performed: differential interference contrast (DIC) microscopy, confocal microscopy, determination of reactive oxygen species (ROS), tunnel assay (apoptosis), fluorescence spectroscopy, transmission spectroscopy, and a trypan blue exclusion dye test.
Human umbilical vein endothelial (HUVEC) cells were cultured at 105 cells/2 mL in six well plates for about 24 hours at 37° C. and 5% CO2 in EBM complete media. For investigating the cellular localization, cells were plated on glass cover slips and grown up to 90% confluence in six well plates and then incubated with EuIII(OH)3 nanorods at a concentration range of 20-100 μg/mL. After 24 hours of incubation, the cover slips were rinsed extensively with phosphate buffer saline, and the cells were fixed with freshly prepared 4% para-formaldehyde in PBS for 15 minutes at room temperature and then re-hydrated with PBS. Once all cells were fixed, the cover slips with the cells were mounted onto glass slides with Fluor Save mounting media and examined using DIC and confocal microscopy. For investigating the formation of reactive oxygen species (ROS), the Image-iT™ LIVE Green Reactive Oxygen Species Detection Kit (Cat. No. #I36007; Molecular Probes, USA) was used according to the manufacturer's instructions with the treated and untreated cells finally mounted onto glass slides with Fluor Save mounting media and examined using confocal microscopy. For a tunnel assay, cells were mounted onto glass slides with mounting media with DAPI (4′-6-Diamidino-2-phenylindole) and examined using confocal microscopy according to the manufacturer's instructions (Roche, Cat. No. # 12 156 792 910).
In another set, HUVEC cells (105 cells/2 mL) were cultured in six well plates and treated with EuIII(OH)3 or TbIII(OH)3 nanorods in EBM complete media without cover slips. After 24 hours of incubation with nanorods, the cells were washed with PBS, trypsinized, and neutralized. The cells were washed by centrifugation, re-suspended in the PBS, and examined using fluorescence spectroscopy and TEM. Cell viability for another set of cells was determined through staining with Trypan Blue, and cells were counted using a hemocytometer.
Cell viability and cell proliferation tests: An in vitro toxicity analysis in terms of inhibition of proliferation using [3H]thymidine incorporation assay to normal endothelial cells (HUVEC) was performed as described elsewhere (Basu et al., Nat Med., 7:569 (2001)). Briefly, endothelial cells (HUVEC; 2×104) were seeded in 24-well plates, cultured for one day in EBM, serum-starved (0.1% serum) for 24 hours, and then treated with different concentrations (0, 20, 50 or 100 μg/mL) of EuIII(OH)3 (
Apoptosis assay: To perform a tunnel apoptosis assay, cells were seeded into 6-well plates at a density of 105 cells/2 mL of medium per well and grown overnight on cover slips. The cells were incubated with EuIII(OH)3 nanorods at different concentrations, mounted onto glass slides with Fluor Save mounting media with DAPI (4′-6-Diamidino-2-phenylindole), and examined using confocal microscopy according to the manufacture's instructions (Roche, USA, Cat. No. # 12 156 792 910). The red colored apoptotic cells were visualized using a microscope, counted (6 fields per sample), and photographed using digital fluorescence camera.
Cell cycle: The cell cycle analysis was performed according to the following standard procedure. DNA content was measured after staining cells with propidium iodide (PI). After treatment of EuIII(OH)3 nanorods, HUVEC cells were washed in PBS (3×) and fixed in 95% ethanol for 1 hour. Cells were re-hydrated, washed in PBS, and treated with RNaseA (1 mg/mL) followed by staining with PI (100 μg/mL). Similar experiments were done with control cells (No EuIII(OH)3 nanorods). Flow cytometric quantification of DNA was done by a FACScan (Becton-Dickinson), and the data analysis was carried out using Modfit software.
Western blot for Map kinase Phosphorylation: Harvested HUVEC cells were washed two times with cold PBS and lysed with ice-cold radioimmunoprecipitation (RIPA) buffer with freshly added 0.01% protease inhibitor cocktail (Sigma). After being incubated on ice for 10 minutes, the cells were centrifuged at 13,000 rpm for 10 minutes at 4° C. After measurement of protein concentration using a photometric method, 20 μg of protein were electrophoresed on a 10% (Tris-HCl) preparative polyacrylamide gel under reducing conditions and transferred to a nitrocellulose membrane by wet blotting. Membranes were cut into strips, blocked in 5% dry milk in tris-buffered saline for 1 hour, incubated overnight with monoclonal mouse IgG (Cat. No # OP72-100UG) for total map kinase or with anti-phospho map kinase (rabbit polyclonal IgG, Cat. No. # 07-467) antibodies, and then with HRP-coupled secondary antibodies (anti-mouse IgG or anti-rabbit IgG-HRP (Cat. #Sc-2301)) at 37° C. for 40 minutes. Detection was performed using a chemiluminescent substrate.
CAM assay: Chick eggs were maintained in a humidified 39° C. incubator (Lyon Electric, Calif.), as described elsewhere (Vlahakis et al., J. Biol. Chem., 282(20):15187-15196 (2007)). Pellets containing 0.5% methylcellulose plus recombinant human VEGF-A (50 ng) or bFGF (150 ng) were placed onto the CAM's of 10-day-old chick pathogen-free embryos (SPAFAS; Charles River Laboratories, Wilmington, Mass.). The CAM's were exposed by cutting a small window in the egg shell to facilitate application of the pellet. Relevant antibodies or agonist/antagonist compounds were applied to the site 24 hours after stimulation with VEGF polypeptides. In some cases, a suspension of europium hydroxide nanorods in Tris-EDTA buffer was applied using a micro-syringe. CAMs were imaged on day 13 either following fixation and excision or with real time live imaging using a digital camera (Canon Supershot6) attached to a Zeiss stereomicroscope. Angiogenesis was quantified by counting branch points arising from tertiary vessels from a minimum of ten specimens from three separate experiments.
The following techniques were performed to characterize EuIII(OH)3 nanorods.
X-ray diffraction (XRD): The structure and phase purity of the as-synthesized samples were determined by X-ray diffraction (XRD) analysis using a Bruker AXS D8 Advance Powder X-ray diffractometer (using CuKαλ=1.5418 Å radiation).
Thermo-gravimetric (TG) and Differential Scanning Calorimetric (DSC)
Analysis: TGA of the as-synthesized sample was carried out under a stream of nitrogen at a heating rate of 10° C./minute from 30° C. to 700° C. using a METTLER TOLEDO TGA/STDA 851. DSC analysis of the as-synthesized sample was carried out on METTLER TOLEDO TC15 using a stream of nitrogen (20 mL/minute) at a heating rate of 4° C./minute in a crimped aluminum crucible from 30° C. to 600° C.
Transmission electron microscopy (TEM) study: The particle morphology (microstructures of the samples) was examined with TEM on a FEI Technai 12 operating at 80 KV. To visualize the internalization of particles inside the cytoplasmic compartment of cells using TEM, procedures described elsewhere were followed (McDowell and Trump, Arch. Path. Lab. Med., 100: 405 (1976) and Spurr, J. Ultrastruct. Res., 26:31 (1969)).
Fluorescence Spectroscopy The excitation and emission (fluorescence) spectra were recorded on Fluorolog-3 Spectrofluorometer (HORIBA JOBINYVON, Longjumeau, France) equipped with xenon lamp as the monochromator excitation source.
Differential interference contrast (DIV) microscopy: After fixation of cells on cover slips, the cells were mounted onto glass slides with Fluor Save mounting media and examined for DIC. Pictures were captured by AXIOCAM high-resolution digital camera using AXIOVER 135 TV microscope (ZEISS, Germany).
Confocal Fluorescence Microscopy for EuIII(OH)3, Tunel assay, and ROS: Two dimensional confocal fluorescence microscopy images were collected through use of LSM 510 confocal laser scan microscope (Carl Zeiss, Inc., Oberkochcn, Germany) with C-Apochromat 63×/NA 1.2 water-immersion lense, in conjunction with an Argon ion laser (488 nm excitation). The fluorescence emissions for EuIII(OH)3 nanorods, untreated cells, and cells treated with EuIII(OH)3 nanorods were collected through a 515 nm long pass filter.
For tunnel assay, after mounting the cells onto glass slides with DAPI, the images were collected through use of LSM 510 confocal laser scan microscope (Carl Zeiss, Inc., Oberkochcn, Germany) with C-Apochromat 63 X/1.2 na water-immersion lense. The fluorescence emissions were collected through a 385-470 nm band pass filter in conjunction with an Argon ion laser excitation of 364 nm for DAPI stained blue nuclei. The fluorescence emissions were collected through a 560-615 nm band pass filter in conjunction with HeNel ion laser excitation of 543 nm for TMR red stained apoptotic nuclei.
For ROS, the images were collected through use of LSM 510 confocal laser scan microscope (Carl Zeiss, Inc., Oberkochcn, Germany) with C-Apochromat 63×/1.2 na water-immersion lense. The green fluorescence (oxidation product of carboxy-H2DCFDA) emissions were collected through a 505-550 nm band pass filter in conjunction with an Argon ion laser excitation of 488 nm. The blue fluorescence emissions for Hoechst 33342 stained blue nuclei were collected through a 385-470 nm band pass filter in conjunction with Argon ion laser excitation of 364 nm.
X-ray Diffraction Studies: The crystal structures of the as-synthesized materials were identified by X-ray diffraction (XRD) analysis (
TGA and DSC: To determine the chemical nature (europium hydroxide or europium oxide) of microwave assisted as-synthesized product (60 minutes of microwave irradiation time), TGA and DSC were performed. A representative TGA-DSC profile for as-synthesized product was obtained (
Similar behaviors also were observed for other as-synthesized products, which were obtained after 5 minutes, 10 minutes, 20 minutes, and 40 minutes of microwave heating. Combination of the results of XRD, DSC, and TGA indicated that the as-synthesized materials were europium(III) hydroxide [Eu(OH)3].
Similarly, thermo gravimetric analysis of other lanthanide hydroxide products (after 60 minutes of microwave irradiation) are presented in
Transmission electron microscopy of nanorods: The morphologies of as-synthesized europium(III) hydroxide [Eu(OH)3] materials obtained after microwave heating at different times were characterized by TEM (
The morphologies of as-synthesized Nd(III) hydroxide [NdIII(OH)3] materials obtained after microwave heating for different times were characterized by TEM (
The morphologies of as-synthesized Sm(III) hydroxide [SmIII(OH)3] materials obtained after microwave heating for different times were characterized by TEM (
The morphologies of as-synthesized Gd(III) hydroxide [GdIII(OH)3] and Tb(III) hydroxide [TbIII(OH)3] materials obtained after 60 minutes of microwave heating were characterized by TEM (
Fluorescence spectroscopy: The excitation and emission spectra of Eu3 ion in EuIII(OH)3 nanorods arose from transitions of electrons within the 4f shells. The fluorescent emission and excitation spectra of europium hydroxide are shown in
To determine if the fluorescence activity of these EuIII(OH)3 nanorods remains unchanged even inside the cells, the emission (fluorescence) spectra of the endothelial cells incubated for 24 hours with these nanorods at various concentrations (5-100 μg/mL) were recorded on a Fluorolog-3 Spectrofluorometer after extensive washing with PBS (phosphate buffer saline). Curves a-, b-, c-, d- and e- of
A number of methods, such as differential interference contrast (DIC) microscopy, confocal microscopy, and transmission electron microscopy (TEM) were used to determine cellular trajectories of nanorods.
DIC: Differential interference contrast (DIC) microscopy pictures (
Confocal microscopy: Fluorescence properties of HUVEC loaded with inorganic fluorescent EuIII(OH)3 nanorods and their corresponding phase images detected by confocal microscopy are presented in the first and second columns of
TEM for nanorods inside the cells: The direct proof of internalization of EuIII(OH)3 nanorods inside the cytoplasmic part of cells was the TEM images of the cells treated with these nanorods at different concentrations.
Taken together, the results from fluorescence spectroscopy, DIC, confocal microscopy, and TEM indicate that these fluorescent nanorods can be internalized in a cell system and readily visualized by microscopy. These nanorods thus constituted interesting fluorescent probes for the targeting of various molecules to specific cells.
Cell proliferation and viability tests: Before using the inorganic nanorods as a fluorescent label into endothelial cells (HUVEC), the viability of HUVEC was tested after treatment with EuIII(OH)3 nanorods at different concentrations from 20-100 μg/mL and incubation for 1-2 days to observe apoptosis. There was no difference of cell deaths between the control cells (no treatment) and cells treated with these nanorods as assessed by Trypan Blue exclusion assay. These results indicated that these nanorods were biocompatible with the cells, as they did not affect the cell viability in 24-48 hours.
The EuIII(OH)3 nanorods' in vitro toxicity was examined in terms of inhibition of proliferation using a [3H] Thymidine incorporation assay (Kang et al., J. Am. Soc. Nephrol., 13:806-816 (2002)) to normal endothelial cells (HUVEC). These nanorods were not toxic to HUVEC (
The NdIII(OH)3 nanorods also were observed to be non-toxic to HUVEC (
The SmIII(OH)3 nanorods also were observed to be non-toxic to HUVEC (
The TbIII(OH)3 nanorods also were observed to be non-toxic to HUVEC (
Apoptosis: According to a tunnel based apoptosis assay, the red colored nuclei were tunnel positive (
Another group (Kirchner et al., Nano. Lett., 5:331 (2005)) indicated that cellular toxicity of stable nanomaterials is primarily due to aggregation rather than the release of Cd elements. The work provided herein, however, uses nanorods of an entirely different material than cadmium-based materials. Thus, the mode of action of EuIII(OH)3 nanorods is likely to be different than Cd-based materials, and that is what was observed.
Cell cycle: To investigate the mechanism of HUVEC cell proliferation in the presence of EuIII(OH)3 nanorods, cell cycle analysis was carried out (
Map kinase phosphorylation: To further confirm the results obtained from the cell proliferation assay and cell cycle analysis, Western blot analyses of control HUVEC cells (untreated) and HUVEC cells treated with EuIII(OH)3 nanorods at a concentration of 50 μg/mL for different times (e.g., 5 minutes to 24 hours) were performed. HUVEC cells were treated with vascular endothelial growth factor (VEGF) at the concentration of 10 ng/mL for 5 minutes in positive control experiments (Bhattacharya et al., Nano Lett., 4(12):2479-2481 (2004)).
Conversely, with increasing the concentration of EuIII(OH)3 nanorods (20-100 μg/mL), map kinase phosphorylation increased, reaching a maximum at 50 μg/mL. Map kinase phosphorylation decreased at 100 μg/mL. These results support the cell proliferation assay results. Therefore, it is concluded that cell proliferation of HUVEC cells after treatment with these nanorods can occur through map kinase phosphorylation pathways.
ROS: There was no green fluorescence (
CAM assay (Nanoparticles induce in vivo angiogenesis): To determine the in vivo relevance of the in vitro findings, chick CAM assays were performed to measure nanoparticle-induced angiogenesis. A control experiment where CAMs were treated with only TE (tris-EDTA) buffer solution was performed (
In summary, europium(III) hydroxide nanorods, which can be used as inorganic fluorescent materials, were synthesized by a microwave technique, which was simple, fast, clean, efficient, economical, non-toxic, and eco-friendly. The europium(III) hydroxide nanorods retained their fluorescent properties even inside endothelial cells (HUVEC). They were characterized by fluorescence spectroscopy, differential interference contrast microscopy (DIC), confocal microscopy, and transmission electron microscopy (TEM). The nanorods have several advantages over traditional organic dyes as fluorescent labels in biology. For example, these nanorods can promote HUVEC cell proliferation, observed by a [3H]thymidine incorporation assay and cell cycle assay. Further, pro-angiogenic properties of Eu(OH)3 nanorods were discovered using a CAM assay, which is well established and widely used as a model to examine angiogenesis and anti-angiogenesis.
The europium hydroxide nanorods provided herein can be used as (a) stable and bright fluorescent labels in biology and medicine, (b) pro-angiogenic materials in in vivo systems, and (c) drug delivery vehicles after being conjugated to a drug molecule. In addition, the non-toxic, europium hydroxide nanorods provided herein can be used on heart or limb ischemic tissues for human beings.
Eighteen nude mice (male) were randomized into three groups of 6 animals per group receiving 0 (control group with Tris-EDTA solution injection), 20 (1 mgKg−1 day−1), or 100 μg (5 mgKg−1 day−1) of europium hydroxide [EuIII(OH)3] nanorods in Tris-EDTA through the IP route of administration for one week. The mice were weighed and examined once per day for any adverse effects or clinical signs throughout the week of regular injections with europium hydroxide nanorods. A mixture of ketamine/xylazine was used to anesthetize mice to facilitate handling. For biochemical and hematological toxicity analysis, blood and serum were collected at the time of sacrifice. Mice in the control groups were sacrificed at the same time as mice of the corresponding experimental group in order to evaluate the effect of the europium hydroxide nanorods in those mice compared to control animals. Mice were sacrificed using the carbon dioxide inhalation method after collection of blood. Hematology analytes included CBC without differential hemoglobin, hematocrit, erythrocytes, mean corpuscular volume (MCV), RBC distribution width, leukocytes and platelet count. Blood chemistry analytes included alkaline phosphates, S (ALP), aspartate aminotransferase (AST), alanine aminotransferase(ALT), creatinine(CR), bilirubin total-S (TBLI), and blood Urea nitrogen (BUN).
In a 7-day toxicity study, intravenous injection of europium hydroxide nanorods (1 mgKg−1 day−1 and 5 mgKg−1 day−1) in Tris-EDTA buffer showed normal hematology (Table 1) and blood chemistry (Table 2). These results indicate that over the above-mentioned dosage, europium hydroxide nanorods appear non-toxic in the in vivo model.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This Application claims priority to U.S. Provisional Application No. 60/837,807, filed Aug. 14, 2006.
This invention was made with government support under grant numbers CA78383 and HL70567 awarded by National Institutes of Health. The government has certain rights in the invention.
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/US2007/075926 | 8/14/2007 | WO | 00 | 6/22/2009 |
Number | Date | Country | |
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60837807 | Aug 2006 | US |