Search

Superparamagnetic relaxometry (SPMR) is a highly sensitive technique for the in vivo detection of tumor cells and may improve early stage detection of cancers. SPMR employs superparamagnetic iron oxide nanoparticles (SPION). After a brief magnetizing pulse is used to align the SPION, SPMR measures the time decay of SPION using super-conducting quantum interference device (SQUID) sensors. Substantial research has been carried out in developing the SQUID hardware and in improving the properties of the SPION. However, little research has been done in the pre-processing of sensor signals and post-processing source modeling in SPMR. In the present study, we illustrate new pre-processing tools that were developed to: (1) remove trials contaminated with artifacts, (2) evaluate and ensure that a single decay process associated with bounded SPION exists in the data, (3) automatically detect and correct flux jumps, and (4) accurately fit the sensor signals with different decay models. Furthermore, we developed an automated approach based on multi-start dipole imaging technique to obtain the locations and magnitudes of multiple magnetic sources, without initial guesses from the users. A regularization process was implemented to solve the ambiguity issue related to the SPMR source variables. A procedure based on reduced chi-square cost-function was introduced to objectively obtain the adequate number of dipoles that describe the data. The new pre-processing tools and multi-start source imaging approach have been successfully evaluated using phantom data. In conclusion, these tools and multi-start source modeling approach substantially enhance the accuracy and sensitivity in detecting and localizing sources from the SPMR signals. Furthermore, multi-start approach with regularization provided robust and accurate solutions for a poor SNR condition similar to the SPMR detection sensitivity in the order of 1000 cells. We believe such algorithms will help establishing the industrial standards for SPMR when applying the technique in pre-clinical and clinical settings.

The Puerto Rican Racer Alsophis portoricensis is known to use venom to subdue lizard prey, and extensive damage to specific lizard body tissues has been well documented. The toxicity and biochemistry of the venom, however, has not been explored extensively. We employed biological assays and proteomic techniques to characterize venom from A. portoricensis anegadae collected from Guana Island, British Virgin Islands. High metalloproteinase and gelatinase, as well as low acetylcholinesterase and phosphodiesterase activities were detected, and the venom hydrolyzed the alpha-subunit of human fibrinogen very rapidly. SDS-PAGE analysis of venoms revealed up to 22 protein bands, with masses of approximately 5-160 kDa; very little variation among individual snakes or within one snake between venom extractions was observed. Most bands were approximately 25-62 kD, but MALDI-TOF analysis of crude venom indicated considerable complexity in the 1.5-13 kD mass range, including low intensity peaks in the 6.2-8.8 kD mass range (potential three-finger toxins). MALDI-TOF/TOF MS analysis of tryptic peptides confirmed that a 25 kDa band was a venom cysteine-rich secretory protein (CRiSP) with sequence homology with tigrin, a CRiSP from the natricine colubrid Rhabdophis tigrinus. The venom was quite toxic to NSA mice (Mus musculus: LD(50)=2.1 microg/g), as well as to Anolis lizards (A. carolinensis: 3.8 microg/g). Histology of the venom gland showed distinctive differences from the supralabial salivary glands (serous vs. mucosecretory), and like the Brown Treesnake (Boiga irregularis), another rear-fanged snake, serous secretory cells are arranged in densely packed secretory tubules, with little venom present in tubule lumina. These results clearly demonstrate that venom from A. portoricensis shares components with venoms of front-fanged snakes as well as with other rear-fanged species. Venom from A. portoricensis, in particular the prominent metalloproteinase activity, likely serves an important trophic function by facilitating prey handling and predigestion of prey.

Superparamagnetic Relaxometry (SPMR) is a non-invasive technique that utilizes superconducting quantum interference device (SQUID) detectors to localize and quantify the magnetization of superparamagnetic iron oxide (Fe3O4) nanoparticles (NPs) specifically bound to cancerous tumors. In an SPMR measurement, polyethylene glycol (PEG) coated NPs are functionalized with a tumor-targeting monoclonal antibody and injected intravenously. NPs that reach and bind to the target tissue are measured by the MRX™ instrument, while unbound nanoparticles, such as those freely circulating in the bloodstream, are not detected. Here, we demonstrate the use of SPMR for specific cancer detection using long-circulating anti-Her2 antibody conjugated PrecisionMRX® NPs in vitro and in vivo. The stability and biofunctionality of conjugated nanoparticles were measured by dynamic light scattering, gel electrophoresis, and ELISA. A cell competition assay was developed to measure specific binding of NPs to Her2 positive (BT474) and Her2 negative (MCF7) cells in vitro. Specificity was defined by the ability of the native antibody to competitively block the binding of the anti-Her2 conjugated NPs to the Her-2 antigen expressed on the cell surface. For in vivo studies, nude mice with xenograft BT474 tumors were intravenously injected with anti-Her2 NPs at a dose of 20 mg/kg of body mass, while control mice were injected with PEG only NPs. Mice were measured individually on the MRX™ instrument at successive time points over the course of 24 hours. At selected intervals during the 24-hour period, blood, tumor, and organs were harvested and analyzed for SPMR signals and anti-Her2 content. In vitro, the anti-Her2 NPs exhibited specific binding to BT474 cells, with little to no binding in MCF7 cells. In vivo, MRX measurements of mice injected with anti-Her2 NPs showed a measurable magnetic signal in the tumor that reached a near maximum approximately four hours after injection. Conversely, mice injected with unconjugated nanoparticles had no measurable tumor uptake. Finally, 24 hours post-injection, 4 – 8% of NPs and anti-Her2 were measurable in the blood, indicating long-term stability of the NP construct in circulation. Together, these results suggest targeted delivery of conjugated NPs to cancerous tissue in vivo and the utility of SPMR for the sensitive and specific detection of cancer in vivo. This work was performed, in part, at the Center for Integrated Nanotechnologies, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000. Citation Format: Erika C. Vreeland, Kayla E. Minser, Caroline L. Weldon, Andrew Gomez, Todor Karaulanov, Helen J. Hathaway, William H. Anderson, Christopher Nettles, Dale L. Huber, Giulio Paciotti. Sensitive, specific detection of Her-2 positive tumors in mice using superparamagnetic relaxometry (SPMR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2859. doi:10.1158/1538-7445.AM2017-2859

Superparamagnetic Relaxometry (SPMR) is combination technology for the early detection of cancer. Conceptually, PEGylated superparamagnetic iron oxide (Fe3O4) nanoparticles (NPs) coupled with a tumor targeting monoclonal antibody are systemically administered and target solid tumors by both passive (EPR) and active (receptor targeting) mechanisms. Once bound to the target cells, the NPs are magnetized by a brief, low field magnetic pulse to create macroscopic magnetization. Once the field is removed, the particle’s magnetization decays and is measurable by superconducting quantum interference device (SQUID) detectors. The pattern of decay, known as Néel relaxation, exhibits a difference in latency specific to bound nanoparticles and differs from the Brownian decay exhibited by free/unbound particles. As a proof of concept, we developed PEGylated NPs that are covalently bound to an anti-Her-2 monoclonal antibody. In vitro, the nanoconstruct exhibits specific binding to the Her-2 overexpressing BT-474 breast cancer cells, with little to no binding to Her-2 negative MCF-7cells. Similar patterns of selective targeting were observed in vivo. To improve the specific detection of SPMR signals in tumors we developed a novel system that selectively enhances the magnetic signal at tumor sites and reduces the contribution of similar signals at off target sites such as the liver and spleen. For these studies, we created phantoms of varying strength to mimic tumor and non-specific signals. We observed that by creating non-homogenous excitation magnetic field patterns, we reduced the contribution of the non-specific signals by an order of magnitude, effectively increasing the signal to noise ratio of the tumor signal. These results are clinically relevant and support the use of SPMR in the detection of small tumors in cancer patients. Citation Format: Todor Karaulanov, Erika C. Vreeland, Andrew Gomez, Kayla E. Minser, Caroline L. Weldon, William H. Anderson, Christopher Nettles, Giulio Paciotti. Enhancing the in vivo detection of cancer by manipulating magnetic fields applied to tumor targeting superparamagnetic iron oxide nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1865. doi:10.1158/1538-7445.AM2017-1865

Current methods for detecting solid tumors lack sensitivity and diagnose primary and metastatic lesions only after the tumor is well established. Superparamagnetic Relaxometry (SPMR) is a combination technology that utilizes superconducting quantum interference detectors (SQUID) to measure the magnetization of superparamagnetic, tumor-targeting magnetite (Fe3O4) nanoparticles. Conceptually, PEGylated Fe3O4 nanoparticles labeled with a tumor targeting moiety (i.e., a monoclonal antibody) are intravenously injected and specifically target solid tumors utilizing both passive (the EPR effect) and active (receptor-mediated) mechanisms. Subsequently, the Fe3O4 nanoparticles are magnetized by a low field magnetic pulse in the MRX™ instrument and only those particles that are bound to their target site are measured by the SQUID sensors. Unbound nanoparticles are not detected. To demonstrate the utility of SPMR in detecting cancer we used PEGylated PrecisionMRX® nanoparticles that are covalently linked with a monoclonal antibody (mAb) targeting ERB-2 (anti-Her2). The particles were characterized for size (by dynamic light scattering), free and bound mAb (by ELISA), antibody potency (by bioassay) and stealth (in plasma interaction studies). In vitro, the anti-Her2 conjugated particles exhibited specific binding to ERB-2 overexpressing breast cancer cells (MCF-7/Her2-18). Specific binding was defined by the ability of the native mAb to competitively block the binding of the anti-HER-2 conjugated particles to ERB-2 antigen coated on ELISA plates or expressed on the cell surface. In addition, in ERB-2 negative cell lines, the anti-Her2 conjugated particles exhibited little to no binding. In vivo, anti-Her2 conjugated PrecisionMRX exhibited significantly longer circulation times when compared to unPEGylated particles. Distinct magnetic dipoles were detected by the MRX instrument at the target site (the tumor) and site of nanoparticle elimination (the liver). These data were confirmed in excised organs showing significant magnetic moments in the liver, tumor, and spleen. Analysis of the MRX SPMR data suggest that the technology can detect as few as 10,000 cancer cells in vivo by optimizing the nanoparticles for stealth and targeting. This work was performed, in part, at the Center for Integrated Nanotechnologies, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000. Citation Format: Weldon CL, Minser KE, Gomez A, Anderson WH, Karaulanov T, Hathaway HJ, Huber DL, Vreeland EC, Paciotti G. Specific detection of anti-Her2 PEGylated PrecisionMRX® nanoparticles measured using superparamagnetic relaxometry [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-01-08.

We report on the use of Superparamagnetic Relaxometry (SPMR) as a technique for monitoring the distribution of superparamagnetic magnetite (Fe3O4) nanoparticles in vivo. SPMR uses superconducting quantum interference device (SQUID) sensors to measure the remnant magnetization of nanoparticles bound to cells following a brief magnetizing pulse. Unbound nanoparticles are not detected, making SPMR uniquely capable of discriminating between nanoparticles that are circulating freely in the bloodstream from those that have been immobilized in organs and tissues throughout the body. The rate of clearance of magnetic nanoparticles from circulation is largely dependent on their hydrodynamic diameter and surface chemistry. In this study, biodistribution of PrecisionMRX™ nanoparticles with 25 nm cores and two different surface coatings were followed using the MRX™ instrument. In independent experiments, negatively charged carboxylate nanoparticles and methoxy-PEG (mPEG) functionalized nanoparticles with a near neutral charge were suspended in 100 μL of saline. Nanoparticle suspensions were injected intravenously via tail vein into Balb/C mice at a dose of 5 mg/kg of body mass, while control mice were injected with 100 μL of saline solution. Mice were measured individually on the MRX instrument at successive time points over the course of 24 hours. At selected intervals during the 24 hour period, mice were euthanized, exsanguinated, and organs harvested for ex vivo measurements on the MRX. Organs and blood were subsequently assayed for iron content. MRX measurements of mice injected with carboxylate nanoparticles showed a strong magnetic signal localized in the region of liver that reached a maximum several minutes after injection. The negative surface charge of the carboxylate nanoparticles likely resulted in their rapid opsonization in the blood and removal from circulation by phagocytic cells in the liver. Conversely, MRX measurements of mice injected with mPEG coated nanoparticles indicated an extended circulation time, with a gradually increasing magnetic signal in the liver that had not yet reached a maximum after four hours. Together, these experiments demonstrate the utility in using SPMR to measure the clearance of nanoparticles from the bloodstream following injection, an important parameter in the design of optimal coatings for in vivo diagnostic and therapeutic use. This work was performed, in part, at the Center for Integrated Nanotechnologies, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000. Citation Format: Andrew Gomez, Kayla E. Minser, Caroline L. Weldon, Bill Anderson, Todor Karaulanov, Helen J. Hathaway, Dale L. Huber, Edward R. Flynn, Erika Vreeland. Monitoring in vivo biodistribution of superparamagnetic nanoparticles using superparamagnetic relaxometry (SPMR). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4239.

Metalloproteinases from snake venoms are often multi-domain enzymes involved in degradation of a variety of structural proteins. Hemorrhage and tissue necrosis are common manifestations of viperid envenomations in humans, largely due to the actions of prominent metalloproteinases, and envenomation by rear-fanged snakes may also cause hemorrhage. We purified the major metalloproteinase in Alsophis portoricensis (Puerto Rican Racer) venom through HPLC size exclusion and ion exchange chromatography. Named alsophinase, it is the first protein purified and characterized from the venom of Alsophis . Alsophinase is a single polypeptide chain protein, and based on mass, activity and complete inhibition by 1,10-phenanthroline, it is a class P-III snake venom member of the M12 ADAM family of metalloproteinases. Alsophinase has a molecular mass of 56.003 kDa and an N-terminal sequence of QDTYLNAKKYIEFYLVVDNGMFxKYSxxFTV, with 67% sequence identity to a metalloproteinase isolated from venom of Philodryas olfersii (another rear-fanged species). Alsophinase rapidly catalyzed cleavage of only the Ala14–Leu15 bond of oxidized insulin B chain, had potent hemorrhagic activity in mice, and degraded only the α-subunit of human fibrinogen in vitro . Alsophinase is responsible for hemorrhagic and fibrinogenolytic activity of crude venom, and it may contribute to localized edema and ecchymosis associated with human envenomations by A. portoricensis . It may be more specific in peptide bond recognition than many well-characterized viperid P-III metalloproteinases, and it could have utility as a new protein fragmentation enzyme for mass spectrometry studies.