Patent Application
20160271275
The present invention relates to bismuth-iron oxide contrast agents, and methods of using the same.
The field of nanoparticle contrast agents for computed tomography (CT) has grown rapidly over the past decade. Compared to clinically available small molecule contrast agents, nanoparticle-based imaging probes have several advantages, such as the ability to carry a higher payload of contrast-producing material and longer circulation half-lives. Nanoparticles can be efficiently targeted using antibodies, proteins, peptides or other targeting ligands, or used in cell tracking. Also, multiple properties can be integrated into nanoparticles, such as various contrast-generating materials that enable multi-modality imaging or a combination of imaging and therapeutics. Lastly, growing concerns over the biocompatibility of iodinated agents, especially in patients with compromised renal function, has motivated the exploration of new CT contrast agent formulations.
Several research groups have evaluated gold nanoparticles as CT contrast agents. Gold nanoparticles produce strong CT contrast (up to twice that of iodine), are highly biocompatible, can be used for vascular imaging and have been used for targeted CT imaging. An advantage of gold nanoparticles is extensive experience with their synthesis, allowing control over their size, morphology and coating. However, few syntheses of bismuth nanoparticles suitable to be contrast agents have been achieved. Thus, there remains a need for bismuth-based nanoparticles that can work safely and effectively for both CT and magnetic resonance imaging (MRI) purposes.
An embodiment of the present invention relates to a nanoparticle for use as a contrast agent, wherein the nanoparticle comprises a core comprising bismuth and iron oxide, and an outer coating surrounding the core. According to preferred embodiments, the outer coating comprises dextran.
Another embodiment of the present invention relates to a contrast agent composition comprising nanoparticles, a carrier, and one or more optional additives, wherein at least some of the nanoparticles (preferably a majority of the nanoparticles, most preferably all of the nanoparticles) each comprises a core comprising bismuth and iron oxide, and an outer coating surrounding the core.
Another embodiment of the present invention relates to a method of using the contrast agent composition comprising administering the contrast agent composition to a subject. Preferably, the method further comprises imaging the subject (e.g., by CT and/or MRI).
Another embodiment of the present invention relates to a method of making nanoparticles comprising co-precipitating iron (preferably ferrous chloride and ferric chloride) and one or more bismuth salts (preferably bismuth nitrate) in the presence of dextran.
Embodiments of the present invention relate to bismuth-iron oxide nanoparticles (BION) that are dextran-coated, wherein the nanoparticles have the advantage of acting as contrast agents for various imaging techniques, including both computed tomography (CT) and magnetic resonance imaging (MRI). Similar to gold, bismuth attenuates X-rays strongly. Additionally, bismuth is inexpensive (about $0.02/g) as compared to gold, and is thought to be a highly biocompatible heavy metal. The dextran-coated BION are preferably biocompatible, biodegradable, possess strong X-ray attenuation properties and can be used as T2-weighted MR contrast agents.
Embodiments of the invention are described in the article by Naha, et al., Dextran coated bismuth-iron oxide nanohybrid contrast agents for computed tomography and magnetic resonance imaging, Journal of Materials Chemistry B, 2014, 2, pp. 8239-8248, which is incorporated by reference herein, in its entirety and for all purposes.
According to particular embodiments, the invention relates to a nanoparticle, wherein the nanoparticle comprises a core comprising bismuth and iron oxide (e.g., Fe3O4), and an outer coating surrounding the core. The outer coating either completely surrounds, or substantially surrounds, the bismuth-iron oxide core. The outer coating may comprise one or more of dextran, carboxydextran, aminated dextran, starch, chitosan, poly(oligo(ethylene glycol) methacrylate-co-methacrylic acid), polyglycidyl methacrylate, poly(vinylalcohol), polyacrylic acid carboxylates, diols, catechols/dopamines, hydroxamic acids, phosphine oxides and silanes. According to preferred embodiments, the outer coating comprises dextran.
According to particular embodiments, the bismuth-iron oxide cores of the nanoparticles comprise between about 1 mole % and about 99 mole % bismuth, or between about 1 mole % and about 80 mole % bismuth, or between about 1 mole % and about 70 mole % bismuth, or between about 5 mole % and about 90 mole % bismuth, or between about 5 mole % and about 80 mole % bismuth, or between about 5 mole % and about 70 mole % bismuth, or between about 5 mole % and about 60 mole % bismuth, or between about 5 mole % and about 50 mole % bismuth, or between about 5 mole % and about 40 mole % bismuth, or between about 5 mole % and about 30 mole % bismuth, or between about 5 mole % and about 25 mole % bismuth. The nanoparticles may be any shape or size, but they preferably have a spherical (or substantially spherical) shape. Regardless of their shape, the nanoparticles preferably have an average hydrodynamic diameter of between about 1 nm and about 500 nm, or between about 30 nm and about 170 nm, or between about 40 nm and about 150 nm, or between about 50 nm and about 150 nm, or between about 75 nm and about 150 nm, or between about 90 nm and about 140 nm.
According to particular embodiments, the dextran coating can be formed from material whose molecular weight is between about 1000 Da and about 2000000 Da, or between about 1500 Da and about 750000 Da, or between about 3500 Da and about 500000 Da, or between about 5000 Da and about 250000 Da, or between about 10000 Da and about 150000 Da, or between about 20000 Da and about 110000 Da. A specific formulation may use dextran of a molecular weight of, for example, about 1000, about 1500, about 3500, about 5000, about 10000, about 20000, about 25000, about 40000, about 60000, about 70000, about 110000, about 150000, about 250000, about 500000, about 750000, about 2000000 Da, or others. The weight % of dextran in the formulation can also vary between about 1% and about 50%, or between about 5% and about 40%, or between about 10% and about 30%, and may take values of, for example, about 5%, about 10%, about 20%, about 30%, about 40% or other values, based on the overall weight of the nanoparticle.
According to particular embodiments, the nanoparticles of the present invention further comprise one or more targeting agents (also known as “targeting ligands”). The targeting agent may be a molecule or a structure that provides targeting of the nanoparticle to a desired organ, tissue or cell. Non-limiting examples of such targeting agents include peptides, antibodies, proteins, nucleic acids, small molecules, etc. The targeting agent(s) are preferably attached to the outer coating for targeted imaging. A nanoparticle comprising one or more targeting agents can be targeted to specific diseased areas of the subject's body.
According to additional embodiments, the invention relates to a contrast agent composition comprising nanoparticles of the present invention, e.g., nanoparticles comprising a bismuth-iron oxide core and an outer coating (e.g., a dextran coating), wherein the nanoparticles are provided in a suitable carrier (preferably a liquid carrier) for administration to a subject. As used herein, the term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which nanoparticles of the present invention are administered to a subject. Such carriers are preferably liquids; for example, saline, citrate buffer, phosphate-buffered saline, HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer or Tris buffer are preferred carrier(s). A contrast agent composition of the present invention may also include one or more additives, such as one or more of the following: wetting agents, emulsifying agents, pH buffering agents, antibacterial agents, antioxidants, chelating agents, etc. According to particular embodiments, a method for making a composition of the present invention comprises combining (e.g., mixing or suspending) the nanoparticles with a carrier and one or more optional additives according to known methods.
The terms “subject” and “patient” are used interchangeably herein and refer to a mammalian individual, such as a mouse, rabbit, or human being. In pre-clinical settings, for example, a contrast agent composition of the present invention may be administered to a mouse or rabbit; in clinical settings, the contrast agent composition is preferably administered to a human being.
According to embodiments of the present invention, a method of using bismuth-iron oxide nanoparticles described herein comprises administering the nanoparticles to a subject. According to additional embodiments, a method of using a contrast agent composition described herein comprises administering the contrast agent composition to a subject. For example, the contrast agent composition may be administered to a subject by injection (i.e., intravenously) or by oral administration. The amount of the contrast agent composition that is administered to the subject can be readily determined by one of ordinary skill in the art. For example, the contrast agent composition can be administered to a subject in an amount of between about 1 mg/kg and about 500 mg/kg, or between about 10 mg/kg and about 350 mg/kg, or between about 50 mg/kg and about 200 mg/kg. According to particular embodiments, the method further comprises imaging the subject, preferably by using computed tomography (CT) and/or magnetic resonance imaging (MRI) according to known methods.
According to particular embodiments, a Molday synthesis for iron oxide nanoparticles is employed to make the bismuth-iron oxide nanoparticles, wherein iron (III) and iron (II) chlorides are co-precipitated using ammonia in the presence of dextran. The protocol may be modified by substituting varying amounts of bismuth (III) for iron (III). A described below, a range of such formulations were synthesized to explore the effect of this substitution on the properties of the nanoparticles.
According to particular embodiments, a method of making nanoparticles comprises co-precipitating iron (e.g., ferrous chloride and ferric chloride) and one or more bismuth salts (e.g., bismuth nitrate) in the presence of dextran. Preferably, the method comprises one or more of the following steps: mixing iron (e.g., ferrous chloride and ferric chloride) and one or more bismuth salts (e.g., bismuth nitrate) with a dextran solution to form a dextran-iron-bismuth solution; adding ammonium hydroxide (e.g., 28-30% or other concentrations) to the dextran-iron-bismuth solution; heating the resulting solution to form a nanoparticle suspension; centrifuging the suspension; and collecting the resulting nanoparticles from the suspension. Preferably, the iron (e.g., ferrous chloride and ferric chloride) is dissolved in water and the bismuth salt(s) (e.g., bismuth nitrate) are dissolved in a polyol, e.g., ethylene glycol, prior to addition to the dextran solution. According to particular embodiments, the method of making nanoparticles further comprises attaching one or more targeting agents onto the coating according to known methods (e.g., one or more targeting agents selected from the group consisting of peptides, antibodies, proteins, nucleic acids, small molecules, and a combination thereof).
For example, the method may comprise one or more of the following steps: dissolving dextran in water to form a dextran solution; removing oxygen from the solution (e.g., by purging the solution with nitrogen gas while stirring); mixing iron (e.g., ferrous chloride and ferric chloride) and one or more bismuth salts (e.g., bismuth nitrate) with the dextran solution (preferably, the iron is dissolved in water and the bismuth salt is dissolved in a polyol, e.g., ethylene glycol, prior to addition to the dextran solution) to form a dextran-iron-bismuth solution; adding ammonium hydroxide (e.g., 28-30%) to the dextran-iron-bismuth solution; heating the resulting solution (e.g., at about 90° C. for about an hour) to form a nanoparticle suspension; stirring the suspension at room temperature (e.g., overnight); centrifuging the suspension (e.g., at 20 k rcf for 30 minutes); collecting the supernatant and concentrating it (e.g., using ultrafiltration tubes) to obtain the nanoparticles; and washing the nanoparticles (e.g., with citrate buffer using 100 kDa MW diafiltration columns).
As shown in Table 1, a range of bismuth-iron oxide nanoparticle (BION) formulations can be synthesized, for example, by substituting different percentages of iron (e.g., ferric chloride) with a bismuth salt (e.g., bismuth (III) nitrate). According to particular embodiments, the amount of bismuth salt (e.g., bismuth nitrate) mixed with the dextran solution (relative to the total amount of bismuth salt, ferrous chloride and ferric chloride) is between about 1 wt % and about 99 wt %, or between about 10 wt % and about 90 wt %, or between about 25 wt % and about 90 wt %, or between about 25 wt % and about 75 wt %, or between about 25 wt % and about 60 wt %, as shown in Table 1. According to particular embodiments, the amount of ferrous chloride plus ferric chloride mixed with the dextran solution (relative to the total amount of bismuth salt, ferrous chloride and ferric chloride) is between about 10 wt % and about 90 wt %, or between about 10 wt % and about 75 wt %, or between about 25 wt % and about 75 wt %, or between about 40 wt % and about 75 wt %, as shown in Table 1.
According to preferred embodiments, the amount of bismuth salt (e.g., bismuth nitrate) mixed with the dextran solution (relative to the total amount of bismuth salt, ferrous chloride and ferric chloride) is between about 25 wt % and about 60 wt % (e.g., as shown in the Bi-30 and Bi-50 formulations described below). According to particular embodiments, it was found that inclusion of bismuth resulted in marked increases in X-ray attenuation, in proportion to the amount of bismuth in the formulation (see, e.g.,
The embodiments of the invention are described above using the term “comprising” and variations thereof. However, it is the intent of the inventors that the term “comprising” may be substituted in any of the embodiments described herein with “consisting of” and “consisting essentially of” without departing from the scope of the invention. Unless specified otherwise, all values provided herein include up to and including the starting points and end points given.
The following examples further illustrate embodiments of the invention and are to be construed as illustrative and not in limitation thereof.
Provided below are the results of synthesis and characterization of several dextran-coated bismuth iron oxide nanoparticle (BION) formulations and in vivo imaging experiments using a selected formulation (Bi-30). BION were synthesized through co-precipitation of ferrous chloride, ferric chloride and bismuth nitrate in the presence of dextran. A range of BION formulations were synthesized by substituting several different percentages of ferric chloride with bismuth (III) nitrate. Each BION formulation was characterized using techniques such as transmission electron microscopy (TEM), dynamic light scattering (DLS), inductively coupled plasma optical emission spectroscopy (ICP-OES), relaxometry, energy dispersive X-ray spectroscopy (EDS) and magnetic hysteresis measurements. The biocompatibility of the nanoparticles was evaluated via in vitro incubation with hepatocytes (Hep G2) and fibroblasts (BJSta). Taken together, the results of these experiments indicated that Bi-30 was the preferred formulation to test in vivo. CT and MR imaging experiments, biodistribution measurements and biodegradation studies were carried out using this formulation in mice, which demonstrated the potential of this agent as a dual CT/MRI contrast agent and that it is degradable and excretable in vivo.
Several different BION formulations were synthesized by co-precipitation of iron and bismuth salts in the presence of dextran using a modified version of a protocol previously reported for the synthesis of dextran coated iron oxide. In this process, 12.5 g of dextran T-10 (Pharmacosmos, Holbaek, Denmark) were dissolved in 25 ml of deionized (DI) water. The resulting solution was placed in an ice bath and was purged with nitrogen gas for 30 minutes while stirred, to completely remove oxygen from the flask. For each formulation, varying amounts of ferric chloride and bismuth nitrate, but a fixed amount of ferrous chloride (all purchased from Sigma-Aldrich, St. Louis, USA), as summarized in Table 1, were added to the dextran solution. As the amount of bismuth nitrate was increased, the amount of ferric chloride used in the reaction was reduced, to maintain the molar ratios between the Fe3+/Bi3+ and Fe2+ ions. The ferric chloride and ferrous chloride were each dissolved in 6.5 ml of DI water whereas the bismuth nitrate was dissolved in 3.5 ml of ethylene glycol prior to addition to the dextran solution (ethylene glycol was also added to the reaction when no bismuth was used). Notably, in the absence of bismuth incorporation, there was no difference found in particle size or transverse (T2) relaxivity on the presence or absence of ethylene glycol. A transmission electron microscopy (TEM) image for Bi-0 formulation synthesized without ethylene glycol is presented in
Fifteen ml of concentrated ammonium hydroxide (28-30%) were added to the dextran-iron-bismuth solution using a syringe pump. The ammonium hydroxide flow rate was set to 0.3 μl/min for the first 2.5 hours and then increased to 0.6, 0.9 and 1.2 μl/min for 1 hour each, consecutively; the remainder of the ammonium hydroxide was added to the dextran solution at a rate of 4 μl/min. After addition of ammonium hydroxide, the nanoparticle suspension was heated to 90° C. for an hour and then stirred at room temperature overnight. The resulting nanoparticle suspension was centrifuged at 20 k rcf for 30 minutes. The supernatant was collected and concentrated to 15 ml using ultrafiltration tubes (molecular weight cut off 100 kDa, Sartorius Stedim Biotech, Germany). The nanoparticles were washed with citrate buffer (0.15 M sodium chloride and 20 mM sodium citrate dehydrate, pH 7.4) using 100 kDa MW diafiltration columns (SPECTRUM, CA, USA) continuously for 16 h using a peristaltic pump. After purification, the resulting BION formulations were stored at 4° C. Several formulations were synthesized and termed as Bi-0 Bi-10, Bi-30, etc., where the number indicates the percentage of ferric chloride replaced with bismuth nitrate, as summarized in Table 1.
The hydrodynamic diameter and zeta potential of each BION formulation were measured using a Nano-ZS 90 (Malvern Instrument, Malvern, UK). Hydrodynamic diameter and zeta potential measurements were performed at 25° C. using 1.5 and 1 ml of diluted BION (10 μl from stock was diluted with 2 ml of freshly filtered (0.2 μm) DI water).
Transmission electron microscopy of each BION formulation was performed using a JEOL 1010 microscope operating at 80 kV. Ten μl of diluted BION (10 μl from stock was diluted with 1 ml of DI water) was dropped onto carbon coated copper grid (FCF-200-Cu, Electron Microscopy Sciences, Hatfield, Pa., USA), and allowed to evaporate before imaging.
Elemental analysis of each BION formulation was performed using energy dispersive X-ray spectroscopy. TEM grids were prepared as described above and the elemental analysis was performed using a JEOL 2010F microscope operating at 80 to 200 kV.
The amount of iron and bismuth present in each BION was determined using inductively coupled plasma optical emission spectroscopy (ICP-OES, Spectro Genesis ICP). In brief, 5, 10 and 25 μl of BION from the stock of each formulation were dissolved in 1 ml of concentrated nitric acid (70%). The final volume of each sample was adjusted to 10 ml using deionized water. Bismuth and iron analytical standards were purchased from Fisher Scientific (Pittsburgh, USA). The concentrations of bismuth and iron were determined for each sample, multiplied by the dilution factor and the concentrations thus obtained were averaged to obtain the final bismuth and iron concentration for each BION formulation.
The T2 relaxivity of each BION formulation was measured using a tabletop Minispec MR (Bruker mq60 MR) relaxometer operating at 1.41 T (60 MHz). T2 values were measured for BION formulation whose iron concentration ranged from 0.1 to 0.6 mM. The transverse relaxivity was calculated from the slope of 1/T2 plotted against iron concentration for each formulation.
The magnetic properties of each BION formulation were determined using an alternating gradient magnetometer (Princeton Instruments Corporation, Princeton, N.J., USA). In brief, 5 μl of BION suspensions (12 mg Fe/ml) were dropped on to 4×4 mm2 cover glass slides and allowed to dry. The magnetic properties of each BION formulation were estimated from their hysteresis curves.
The CT phantom was constructed similarly to a described previously protocol. In brief, the concentration of each BION formulation was adjusted to 9.37 mg Fe/ml, whereas the bismuth concentration varied in each formulation (the concentrations of bismuth in Bi-0, Bi-10, Bi-30 and Bi-50 were 0, 1.98, 6.34 and 6.38 mg/ml respectively). These samples were prepared in triplicate in 1.5 ml centrifuge tubes. The tubes were placed in a rack, which was covered in parafilm and the rack placed in a plastic container (24 cm in width) that was filled with water up to 21 cm in height, to simulate the attenuation effects of the abdomen of a patient. The phantom was scanned using a Siemens Definition DS 64-slice clinical CT scanner at 80 kV (550 mA), 100 kV (440 mA), 120 kV (352 mA) and 140 kV (308 mA) with a matrix size of 512×512, field of view 37×37 cm, reconstruction kernel B30f and slice thickness of 0.6 cm. Images were analyzed using Osirix 64 bit (v3.7.1). The attenuation value, in Hounsfield Units (HU) for each sample tube was determined from three different slices and averaged for each sample.
BION samples at a concentration of 0.195 mg Fe/ml were prepared in triplicate in 1.5 ml centrifuge tubes. The tubes were placed in a rack, which was then placed in a hot solution of 2% agarose and 0.35 mM manganese chloride solution. The phantom was cooled to 4° C. to allow the agarose to solidify. The BION phantom was scanned using a 3T MRI system (Tim Trio Model, Siemens Healthcare, Erlangen, Germany). We computed T2 using a spin echo pulse sequence. The scanning parameters used were: echo time (TE)=5.8 ms, repetition time (TR)=10 sec, slice thickness=3 mm, flip angle (FA)=180 degrees, acquisition matrix 184×256, field of view (FOV) 165×230. The MR images of BION phantom were analyzed using Osirix v.3.0.1 32-bit.
Hep G2 (human hepatocellular liver carcinoma) and BJ5ta (human foreskin fibroblast) cells were purchased from ATCC (Manassas, Va., USA). The Hep G2 cells were maintained in Eagle's Minimum Essential Medium, 10% fetal bovine serum (Gibco, Grand Island, N.Y. USA), 45 IU/ml penicillin and 45 IU/ml streptomycin (Gibco). The BJ5ta cells were maintained in a culture medium containing 4 parts of Dulbecco's Modified Eagle's Medium (ATCC), 1 parts of medium 199 (ATCC), 10% fetal bovine serum (Gibco) and 0.01 mg/ml of hygromycin B (Sigma-Aldrich). The cells were grown at 37° C. in 5% CO2 humidified incubator.
In vitro cytotoxicity of each BION formulation was examined in Hep G2 and BJ5ta cells using the MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay (CellTiter 96 cell proliferation assay kit; Promega, Madison, Wis., USA). The assay was performed according to the manufacturer instructions and as reported elsewhere.
In brief, the assay was performed in a 96 well flat bottom micro plate with 10,000 cells seeded in each well. Three different concentrations (i.e., 10, 50 and 100 μg Fe/ml) of each BION formulation were tested in both cell lines. Six replicate wells were used for each control and test concentration per microplate. After 24 hours incubation with BION, the cell monolayer was rinsed gently with sterile PBS. 20 μl of MTS/phenazine methosulfate (PMS) solution and 100 μl of cell culture medium were added to each well and the plates subsequently incubated at 37° C. for 1 hour. After this incubation, the absorbance was measured at 490 nm using a micro plate reader. Three independent experiments were performed for each exposure dose and each BION formulation. The percentage of relative cell viability was calculated and the data presented as mean±standard deviation (n=3).
All animal experiments were performed using wild-type C57BL/6J inbred mice (Jackson Laboratory, Bar Harbor, Me., USA). All in vivo experiments were performed with Bi-30 at a dose of 350 mg Bi/kg body weight, administered via the tail vein.
For CT imaging experiments, pre and post-injection CT images of mice was acquired using a MicroCAT II (Imtek, Inc, Knoxville, Tenn., USA). 8 week old wild-type C57BL/6J mice (n=3) were anesthetized via isoflurane prior to a pre-injection CT scan and then injected with BION (Bi-30). After this injection, mice were scanned at different time intervals post-injection i.e. 5, 30, 60 and 120 minutes. CT images were acquired using the following parameters: slice thickness 100 μm, field of view 51.2 mm×76.8 mm, tube voltage 80 kV and tube current was 500 μA. The reconstruction kernel used a Feldkamp cone beam correction and Shepp-Logan filter. The CT images were analyzed using Osirix 64 bit (v3.7.1). The attenuation values in Hounsfield Units (HU) for several organs (heart, liver, spleen and bladder) were recorded from three different slices and averaged. The data presented is the relative attenuation compared to pre-injection scan (mean±SD).
For MR imaging experiments, pre and post-injection MR images of mice were acquired using a 9.4 T magnet interfaced to a Varian INOVA console (Varian Medical Systems, Palo Alto, Calif., USA). In brief, 8-week-old wild-type C57BL/6J mice (n=3) were anesthetized with isoflurane prior to the pre-injection MR scan. The mice were scanned again two hours after injection with BION (Bi-30). MR images were acquired using the following parameters: TR=200 ms, TE=5 ms, flip angle 20°, number of acquisitions 8 and slice thickness 0.5 mm. The images were analyzed using ImageJ. For each mouse, three different axial slices were chosen for analysis from both the pre and post-injection scans. The signal to noise ratio (SNR) of the liver and adjacent muscle in each slice was calculated by dividing the standard deviation of the background noise. The data is presented as the mean±SD.
The biodistribution of BION (Bi-30) was investigated in 8-week-old wild-type C57BL/6J mice (n=4). A control group of mice (n=4) was employed for estimation of iron content in different organs. At 2 hours post injection of Bi-30, the mice were sacrificed and perfused via the left ventricle with 20 ml PBS. After perfusion, the liver, lungs, heart, spleen and kidneys were harvested. These organs were also harvested in the same way as described above for the control group. All the organs were weighed, diced into small pieces and digested in 800 μl of concentrated nitric acid at 75° C. for nearly 16 hours. Urine samples were collected from the treated (after 2 hour post injection) and control group. The bismuth and iron content were determined using the ICP-OES. Data are presented as mean±SD (n=4).
Samples of the Bi-30 formulation (4 mg Fe) were suspended in either 1 ml of 10% fetal bovine serum (FBS) (in PBS, pH 7.4) or 20 mM citrate buffer (pH 5.5) in 1.5 ml centrifuge tubes and incubated at 37° C. in an oven. At different time intervals (such as 1, 2, 4, 6, 24, 48, 72, 96, 120, 144 and 160 hours) the supernatant from each sample was collected (after centrifugation at 20800 rcf for 20 min) and nanoparticle pellets were resuspended with fresh buffer. Then the tubes were incubated in the oven for next round of sample collection. The released amount of bismuth and iron were analyzed in the samples using ICP-OES.
Dextran coated BION formulations were synthesized by co-precipitation of Fe(III), Fe(II) and Bi(III) in the presence of dextran via the addition of ammonium hydroxide (
The hydrodynamic diameter of the BION formulations increased with increasing amounts of bismuth used in the synthesis (Table 2), even though the cores formed did not increase in size, as evidenced by the TEM images (
The relationship between T2 relaxivity and the amount of bismuth used in synthesis is shown in
BION Phantom Imaging with CT and MRI.
CT images, acquired at 140 kV, of a phantom containing several BION formulations at the same iron concentration (9.4 mg/ml) is presented in
A magnetic resonance image of a phantom containing different BION formulations is shown in
The results of cell viability measurements for BION formulations incubated with BJ5ta or Hep G2 cells are presented in
We studied the CT contrast generation of Bi-30 in wild type mice using a microCT scanner operating at 80 kV. Mice were injected via the tail vein at a dose of 350 mg Bi/kg, similar to the doses of iodinated CT contrast agents used in patients. Representative pre- and post-injection CT images are presented in
MR images of a mouse liver before and after Bi-30 injection are presented in
Our observations of CT contrast in the bladder motivated investigation of biodistribution at 2 hours post-BION injection, to determine whether bismuth and iron were being excreted via this route. This data is presented in
To probe the biodegradation of Bi-30, we incubated samples in 10% FBS at 37° C. After 0, 1 and 24 hours of incubation, the hydrodynamic diameter was measured. The hydrodynamic diameter decreased over time from 98 to 90 nm after one hour and to 48 nm after 24 hours (
In this study, the characterization results suggested that the bismuth is integrated into the iron oxide core. Strong X-ray attenuation was observed from the composite nanohybrid. The magnetic properties and transverse relaxation of the BION formulations were decreased due to the presence of bismuth in the nanoparticle core. In vitro cell viability experiments demonstrated the biocompatibility of the BION. In vivo CT imaging revealed strong X-ray attenuation in the heart and blood vessels over a sustained period. We observed strong contrast in mouse liver post-injection during in vivo MR imaging experiments. The biodistribution and in vitro degradation experiments suggests that the nanoparticles degrade over time and excreted in urine. In summary, dextran coated BION are biocompatible, biodegradable, possess strong X-ray attenuation at CT and also perform as T2-weighted MR contrast agents. Therefore, BION can be used as a dual imaging probe for both CT and MRI.
The following references are incorporated by reference herein, in their entireties and for all purposes:
This application claims priority to U.S. Provisional Application No. 62/133,553, entitled BISMUTH-IRON OXIDE CONTRAST AGENTS, filed Mar. 16, 2015, the contents of which are incorporated by reference herein in their entirety.
This invention was made with government support under Grant No. NIBIB R00EB012165 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62133553 | Mar 2015 | US |
