Synthesis and targeting of gold-coated 177Lu-containing lanthanide phosphate nanoparticles-A potential theranostic agent for pulmonary metastatic disease.

Targeted radiotherapies maximize cytotoxicity to cancer cells. In this work, we describe the synthesis, characterization, and biodistribution of antibody conjugated gold-coated lanthanide phosphate nanoparticles containing 177Lu. [177Lu]Lu0.5Gd0.5(PO4)@Au@PEG800@Ab nanoparticles combine the radiation resistance of crystalline lanthanide phosphate for stability, the magnetic properties of gadolinium for facile separations, and a gold coating that can be readily functionalized for the attachment of targeting moieties. In contrast to current targeted radiotherapeutic pharmaceuticals, the nanoparticle-antibody conjugate can target and deliver multiple beta radiations to a single biologically relevant receptor. Up to 95% of the injected dose was delivered to the lungs using the monoclonal antibody mAb-201b to target the nanoparticles to thrombomodulin receptors. The 208 keV gamma ray from 177Lu decay (11%) can be used for SPECT imaging of the radiotherapeutic agent, while the moderate energy beta emitted in the decay can be highly effective in treating metastatic disease.

LnPO4 nanoparticles doped with Ac-225 and sequestered daughters for targeted alpha therapy.

For targeted alpha therapy (TAT) with 225Ac, daughter radioisotopes from the parent emissions should be controlled. Here, we report on a second-generation layered nanoparticle (NP) with improved daughter retention that can mediate TAT of lung tumor colonies. NPs of La3+, Gd3+, and 225Ac3+ ions were coated with additional layers of GdPO4 and then coated with gold via citrate reduction of NaAuCl4. MAb 201b, targeting thrombomodulin in lung endothelium, was added to a polyethylene glycol (dPEG)-COOH linker. The NPs:mAb ratio was quantified by labeling the mAb with 125I. NPs showed 30% injected dose/organ antibody-mediated uptake in the lung, which increased to 47% in mice pretreated with clodronate liposomes to reduce phagocytosis. Retention of daughter 213Bi in lung tissue was more than 70% at one hour and about 90% at 24 hours postinjection. Treatment of mice with lung-targeted 225Ac NP reduced EMT-6 lung colonies relative to cold antibody competition for targeting or phosphate-buffered saline injected controls. We conclude that LnPO4 NPs represent a viable solution to deliver the 225Ac as an in vivo α generator. The NPs successfully retain a large percentage of the daughter products without compromising the tumoricidal properties of the α-radiation.

Mass spectrometry MALDI imaging of colon cancer biomarkers: a new diagnostic paradigm.

Colorectal cancer (CRC), is the second-leading cause of cancer-related deaths in the USA, affecting both men and women. Current projections show little or no change since the publication of a morbidity and mortality study in 2005. The projected number of new cases for 2008 is 154,000, and the projected number of CRC cancer deaths for 2008 is 53,000. The standard diagnostic paradigm is based on histopathology of either biopsy or surgical specimens. This article suggests a new paradigm for colon cancer diagnosis and staging using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS or IMS). IMS may identify potential tumors in normal tissue of cancer patients and predict those cancer patients who are at risk for recurrent cancer.

Micropipette tips--the unsung heroes of mass spectrometry.

Micropipette tip (tip) pipetting accuracy and precision are functions of tip manufacture consistency, morphological variation, and retained inner tip wall particulate matter. Irregular tip inner wall surfaces and retained particulate matter cause pipetting inaccuracy. Washed and unwashed tips from seven manufacturers were compared using weight by difference (six), microscopic Luxol Fast Blue staining (seven), and matrix-assisted laser desorption/onization time-of-flight (MALDI-TOF) mass spectrometry (six). Photomicrographs revealed tip irregularity and inner wall retained particulate matter. Computer model analysis was used to identify tip irregularity and retained matter. These tests established the utility of a pipette solvent wash to increase the performance and the accuracy of tips and, thus, improve the MALDI mass spectra obtained.

Direct identification of proteins from T47D cells and murine brain tissue by matrix-assisted laser desorption/ionization post-source decay/collision-induced dissociation.

The purpose of this study is to determine the feasibility of the direct matrix-assisted laser desorption/ionization (MALDI) identification of proteins in fixed T47D breast cancer cells and murine brain tissues. The ability to identify proteins from cells and tissue may lead to biomarkers that effectively predict the onset of defined disease states, and their dynamic behavior could be an important hint for drug target discoveries. Direct tissue application of trypsin allows protein identification in cells and tissues, while maintaining spatial integrity and intracellular organization. Using a chemical printer, matrix was co-registered on trypsinized human T47D breast cancer cells and cryo-preserved sections of murine brain tissue, followed by MALDI post-source decay (PSD) or MALDI collision-induced dissociation (CID), respectively. Mass-to-charge (m/z) data from the cells and brain tissues were processed using Mascot software interrogation of the National Center for Biotechnology Information (NCBI) database. Histone H2B was identified from cultured T47D human breast cancer cells. Tubulin beta2 was identified from mouse brain cortex following an induced stroke. These results suggest that MALDI PSD/CID, combined with bioinformatics, can be used for the direct identification of proteins from cells and tissues. Refinements in preparation techniques may improve this approach to provide a tool for quantitative proteomics and clinical analysis.

Minimally invasive method for murine brain fixation.

Complete brain fixation can be achieved with transthoracic cardiac infusion without thoracotomy. Light and electron microscopy tissue sections reveal preservation of cytoplasmic and nuclear structure at all magnification levels. Punched samples were obtained from the fixed tissue specimens in precisely localized areas for study using electron microscopy. This perfusion fixation technique provides both faster tissue harvesting capability and higher quality tissue preservation, without the artifacts of brain swelling and ventricular dilation observed in direct cardiac perfusion. Acute, discrete change in brain tissue can be studied.

A murine photochemical stroke model with histologic correlates of apoptotic and nonapoptotic mechanisms.

INTRODUCTION: The neuronal cell death that occurs after ischemia-induced cerebral infarction (stroke) contains elements of apoptosis and necrosis, an intermediary form of the two, and a distinct excitotoxic process. We previously developed a photochemical model of stroke in the rat. We have now adapted this model for use in the mouse. The present manuscript describes the mouse model. METHODS: Minimal beam intensity (0.1 W/cm(2)) cold white light (8 min exposure) was used to evoke discrete infarcts in the parietal lobes of 11 mice sensitized by the administration of fresh Rose Bengal (10 mg/kg by rapid iv infusion). RESULTS: At 2 h, five out of five mice and at 6 h, six out of six mice demonstrated light microscopic histologic features like those in the rat model. These included a superior ischemic zone with shrunken and pyknotic nuclei, a middle transition zone of edematous vacuolated neuropil but normal neurons with open chromatin and retained Nissl granules, and an inferior zone with normal neurons. There was widespread nuclear terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) in the superior infarct zone in 11/11 mice. However, in the edematous vacuolated transition zone, 11/11 mice had TUNEL positive and negative nuclei randomly mixed. Light microscopic analysis of that same transition zone showed no pyknosis or chromatin bodies in the TUNEL positive or negative cells. DISCUSSION: In mice, photoactivation of Rose Bengal evoked similar infarct and transition zone patterns found previously in rats, with TUNEL evidence of apoptotic and nonapoptotic events. Thus, it will be possible to use this model for further quantitative study of apoptotic and excitotoxic events in wild and transgenic mice.