The assignee for this patent application is
Reporters obtained the following quote from the background information supplied by the inventors: "Magnetic resonance imaging (MRI) has become a powerful technique in the clinical diagnosis of disease and in animal imaging..sup.1-4 MRI is capable of obtaining tomographic images of living subjects with high spatial resolution. It is based on the interaction of water protons with surrounding molecules within tissues in the presence of an external magnetic field..sup.5-8 MR contrast agents.sup.9-11 typically enhance contrast for more accurate diagnosis. Most recently, MR agents have been modified to allow for targeting imaging by conjugating targeting ligand (e.g. antibody, peptide) is conjugated to MR contrast agent..sup.12,13 Among these probes, superparamagnetic nanoparticles.sup.14-16 and paramagnetic metal chelates.sup.8 are the most commonly used..sup.17-21 Superparamagnetic nanoparticles are typically composed of an iron oxide nanoparticle (IONP) surrounded by a polymeric coating to facilitate increased stability in aqueous media..sup.22 They work by shortening the traverse relaxation time (T.sub.2) of surrounding water protons, resulting in a decrease of the signal (negative contrast, dark signal) using the T.sub.2-weighted sequences for the MR scanner..sup.23-28 On the other hand, paramagnetic gadolinium chelates create an increase in signal intensity on T.sub.1-weighted images (positive contrast, bright signal) by shortening the longitudinal relaxation time (T.sub.1) of surrounding water protons..sup.10,17,29-38
"The development of an activatable MR imaging agent that reports on a biological process associated with diseases would greatly advance medical imaging of disease at a molecular level..sup.39-41 Activatable T.sub.1 or T.sub.2 agents, those that results in modulation of either the T.sub.1 or T.sub.2 relaxation time upon target binding, enzymatic activity or biological process associated with disease would be attractive MR imaging agents, resulting in high sensitivity and high signal to noise ratios with low background..sup.20,42-48 Activatable Gd-based T.sub.1 agents have been previously described.sup.8,49,50 and include those designed to be biologically activated by an enzyme such as .beta.-Galactosidase.sup.51,52 and .beta.-Glucoronidase.sup.42,44 as well as those activated by a release of a drug..sup.53,54 Activatable T.sub.2 IONP based agents are less common as it is often difficult to 'quench' the strong superparamagnetic nature of these nanoparticles..sup.26-29,55 Magnetic relaxation switches, have been developed based on IONP that cluster in the presence of a target or enzymatic activity leading to detectable changes in the T.sub.2 relaxation times..sup.56-59 However, the use of these T.sub.2 activatable agents has been difficult to implement in cells or animal studies and it has been limited to their use as nanosensors in molecular diagnostic applications..sup.57,60
"An activatable T.sub.1 agent, one that can induce a faster T.sub.1 relaxation, would result in an increase in the T.sub.1-weighted MR signal intensity upon target recognition for better diagnosis. Such an activatable agent could be beneficial in cancer diagnosis if it were designed to become activated upon tumor targeting, resulting in a brighter signal."
In addition to obtaining background information on this patent application, VerticalNews editors also obtained the inventors' summary information for this patent application: "In accordance with an aspect, there is now described the design, synthesis and characterization of a novel probe that becomes activated in an environment having a less than normal physiological pH, resulting in an increase in the T.sub.1-weighted signal (brighter contrast). In one aspect, the designed probe is composed of a superparamagnetic core, such as an iron oxide nanoparticle, that encapsulates a paramagnetic agent, such as a gadolinium and diethylenetriaminepentacetate (Gd-DTPA) chelate, within hydrophobic pockets of the nanoparticle's polymeric matrix, e.g., a polyacrylic acid (PAA) coating (IO-PAA-Gd-DTPA). While not wishing to be bound by theory, it is believed that the strong magnetic field of the superparamagnetic iron oxide core will affect the relaxation process of the much weaker paramagnetic Gd-DTPA, resulting in quenching of its T.sub.1 signal (FIG. 1). The present inventors observed, for example, that the T.sub.1 relaxation rate (1/T.sub.1) of the Gd(III)-DTPA complex was quenched (OFF/Dark) when the Gd-DTPA complex was encapsulated within the PAA coating of the iron oxide nanoparticle (IO-PAA). Upon release of the quenched Gd-DTPA, an increase in the T.sub.1 relaxation rate was observed with marginal increase in the T.sub.2 relaxation rate (1/T.sub.2). This quenching effect was not observed when the Gd chelate was attached to the surface of the IONP or when a non-magnetic nanoparticles, such as cerium oxide nanoparticles, were used to encapsulate the Gd-DTPA. Corresponding R.sub.1 and R.sub.2 values for the IO-PAA-Gd-DTPA nanocomposite at different pH revealed a pH-dependent increase in the R.sub.1 of the nanocomposite suspension as the pH decreases, indicating T.sub.1 activation at acidic pH. The observed pH dependent increase in R.sub.1 was only observed when Gd-DTPA was encapsulated within the polymeric coating of the nanoparticle, but not when Gd-DTPA was directly attached on the surface of the nanoparticle's polymeric coating.
"In addition, the present inventors have found that the superparamagnetic iron oxide nanocrystal acted as a magnetic quencher for the Gd-DTPA T.sub.1 only when the Gd-DTPA is encapsulated within the nanoparticle's polymeric coating in close proximity to the superparamagnetic core. Also, it was confirmed that the T.sub.2 activation of the probes was not quenched upon encapsulation of Gd-DTPA complex. Furthermore, when the IO-PAA-Gd-DTPA nanocomposite was conjugated with a targeting agent, such as folic acid, its selective internalization and lysosomal localization within folate receptor positive cells allow for selective activation due to the lysosome's acidic pH. Still further, when the folate receptor targeting nanocomposite was used to co-encapsulate a cytotoxic drug (e.g., Taxol), dual delivery of the drug and T.sub.1 imaging activation was achieved. Taken together, the newly developed activatable probes (IO-PAA-Gd-DTPA) combine features of several important modalities, such as: (i) activatable T1-weighted MRI contrast, (ii) T.sub.2-weighted MRI contrast, (iii) receptor-targeted internalization, (iv) biodegradable and biocompatible and/or (v) tumor delivery of anticancer drug(s). These features render the described probes as particularly suitable MR-activatable agents for cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
"FIG. 1 is a schematic representation of the acidic pH-mediated activation of the activatable composite magnetic nanoprobe IO-PAA-Gd-DTPA and corresponding T.sub.1-MR activation.
"FIGS. 2A-2D show the measurement of hydrodynamic diameter by dynamic light scattering (DLS) and the overall size by scanning transmittance electron microscopy (STEM, scale bar 200 nm, Inset) of A) the control probe (IO-PAA) and B) the activatable probe (IO-PAA-Gd-DTPA). C) FT-IR spectra showing successful PAA coating, whereas D) the overall surface charge (zeta potential) of different functional magnetic nanoprobes (carboxylated nanoprobe: -41 mV, alkynated nanoprobe: -16 mV and folate nanoprobe: -29 mV) were measured using zeta seizer, indicating successful surface functionalization of our magnetic nanoprobes.
"FIG. 3 is a schematic representation of the acid-mediated magnetic relaxations of the composite nanoceria NC-PAA-Gd-DTPA nanoprobe and the change in magnetic relaxations was shown by the corresponding T.sub.1-weighted MRI (B=4.7 T) images. DLS and ICP-MS of the nanoprobe aqueous suspension indicated the presence of 88.+-.1 nm nanoparticles with a Gd concentration of 0.315 mg/mL.
"FIGS. 4A-4F show an assessment of magnetic relaxations of activatable magnetic nanoprobe IO-PAA-Gd-DTPA using bench-top magnetic relaxometer (
"FIGS. 5A-5F show an assessment of magnetic relaxations of control magnetic nanoprobe IO-PAA using bench-top magnetic relaxometer (
"FIGS. 6A-6F show an assessment of magnetic relaxations of composite nanoceria NC-PAA-Gd-DTPA using bench-top magnetic relaxometer (
"FIG. 7 is a schematic representation of the Gd-DTPA surface conjugating IO-PAA magnetic nanoprobe, IO-PAA-Gd-DTPA-Surface and the corresponding changes in acid-mediated magnetic relaxations of the Gd-DTPA surface conjugating IO-PAA magnetic nanoprobe, as shown by the T.sub.1- and T.sub.2-weighted MRI (B=4.7 T) images.
"FIGS. 8A-8F show an assessment of magnetic relaxations of nanoceria NC-PAA using bench-top magnetic relaxometer (
"FIGS. 9A-9F show an assessment of magnetic relaxations of Gd-DTPA surface conjugating IO-PAA magnetic nanoprobes, using bench-top magnetic relaxometer (
"FIG. 10A-10D show Magnetic Resonance Imaging (MRI) studies measuring the magnetic activations (T.sub.1- and T.sub.2-maps) of activatable magnetic IO-PAA-Gd-DTPA nanoprobes in
"FIGS. 11A-11D show Magnetic Resonance Imaging (MRI) studies measuring the magnetic activations (using T.sub.1- and T.sub.2-maps) of control magnetic IO-PAA nanoprobes in
"FIG. 12A-12D show intracellular magnetic activations of our folate-decorated activatable IO-PAA-Gd-DTPA-Fol nanoprobe ( , 100 .mu.L, 28 mM) and the control IO-PAA-Fol nanoprobe (.box-solid., 100 .mu.L, 28 mM) using FR-expressing HeLa cells (A and B) and FR-negative H9c2 cells (C and D). Significant activation in inverse spin-lattice magnetic relaxations (1/T.sub.1) was observed from HeLa cells incubated with the activatable IO-PAA-Gd-DTPA-Fol nanoprobes ( , FIG. 12A). As expected, no significant changes in 1/T.sub.2 were observed from HeLa cells due to absence of any T2 activations (FIG. 12B). Neither 1/T.sub.1 (FIG. 12C) nor 1/T2 (FIG. 12D) activations were observed from H9c2 cells due to lack of any receptor-mediated internalizations.
"FIGS. 13A-13B show the rate of release of taxol and Gd-DTPA at 37.degree. C. A) HPLC experiment (.lamda..sub.abs=227 nm) indicated the time-dependent release of taxol from the activatable IO-PAA-Gd-DTPA nanoprobes (50 .mu.L, 28 mM) when incubated at pH=5.0 (.tangle-solidup.) solution. No significant release of taxol was observed (.box-solid.) when incubated in
"FIGS. 14A-14B show the low-pH mediated magnetic activation corroborated the rate of encapsulated drug release at 37.degree. C. A) HPLC experiment (.lamda..sub.abs=227 nm) indicated the time-dependent release of taxol from the activatable IO-PAA-Gd-DTPA nanoprobes (50 .mu.L, 28 mM) when incubated in acidic
"FIGS. 15A-15B show time-dependent in vitro MTT assays for the determination of cytotoxicity of the functional magnetic nanoprobes (1-5, 35 .mu.L, 28 mM in
"FIG. 16 shows the structure of an IO-PAA-Doxorubicin-S-S-Gd DTPA nanoprobe in accordance with an aspect of the present invention.
"FIGS. 17A-F show the assessment of magnetic relaxations of activatable magnetic nanoprobe IO-PAA-Doxorubicin-S-S-Gd-DTPA using bench-top magnetic relaxometer (
For more information, see this patent application: Perez,
Keywords for this news article include: Protons, Hela Cells, Electrolytes, Nanoparticle, Nanotechnology, Tumor Cell Line, Magnetic Resonance, Monovalent Cations, Inorganic Chemicals, Emerging Technologies,
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