Magnetofluorescent Nanoparticles Research Articles

Prostate Cancer-Targeted Imaging Using Magnetofluorescent Polymeric Nanoparticles Functionalized with Bombesin

In this work, the aim was to prepare and characterize a magnetofluorescent polymeric nanoparticle for prostate cancer imaging in vivo. Glycol chitosan (GC) was chemically modified with N-acetyl histidine (NAHis) as a hydrophobic moiety, and bombesin (BBN) was conjugated to the hydrophobically modified GC for use in targeting gastric-releasing peptide receptors (GRPR) overexpressed in prostate cancer cells. NAHis-GC conjugates were labeled with the near-infrared (NIR) fluorophore Cy5.5 (C-NAHis-GC conjugate). BBN-conjugated C-NAHis-GC nanoparticles (BC-NAHis-GC nanoparticles) showed significantly higher binding to the PC3 cell surface than nanoparticles without BBN, and the cellular binding was clearly inhibited by BBN. The tumor-to-muscle ratios of C- and BC-NAHis-GC nanoparticles were 2.26 +/- 0.66 and 5.37 +/- 0.43, respectively. The tumor accumulation of BC-NAHis-GC nanoparticles was clearly reduced by co-injection of BBN. Further, iron oxide nanoparticles (IO) were loaded into BC-NAHis-GC nanoparticles to investigate the possibility of use as a probe for MRI. IO-BC-NAHis-GC nanoparticles were well observed in the PC3 cells, and the blocking with BBN significantly reduced the cellular binding of the nanoparticles. These results demonstrate that the BBN conjugation to NAHis-GC nanoparticles improves their tumor accumulation in PC3-bearing mice in comparison to nanoparticles without BBN, suggesting that BC-NAHis-GC nanoparticles may be useful for prostate cancer imaging.

Towards Ideal Magnetofluorescent Nanoparticles for Bimodal Detection of Breast‐Cancer Cells

An increasing number of novel molecular markers based on nanomaterials for tumor diagnostics have been developed in recent years. Many efforts have focused on the achievement of site-targeted bioconjugated nanoparticles. In contrast, the mechanisms of toxicity, endocytosis, and degradation pathways are still poorly understood, despite their primary importance for clinical translation. In this study, three different model nanoscale magnetofluorescent particle systems (MFNs) are designed and fabricated. These nanoparticles are evaluated in terms of size, morphology, zeta potential, fluorescence efficiency, capability of enhancing T(2) relaxivity of water protons, and stability. Accordingly, two are developed and the mechanism of internalization, the intracellular fate, and the toxicity in MCF-7 adenocarcinoma cells are studied. Besides the well-documented size effect, the anionic charge seems to be a crucial factor for particle internalization, as MFN penetration through the cell membrane could be modulated by surface charge. Ultrastructural analysis of transmission electron micrographs combined with evidence from confocal microscopy reveals that MFNs are internalized by clathrin-mediated endocytosis and macropinocytosis. Moreover, MFNs are found in EEA1-positive endosomes and in lysosomes, indicating that they follow a physiological pathway of endocytosis. Magnetorelaxometric analysis demonstrates that MFNs enable the detection of 5 x 10(5) cells mL(-1) after treatment with particle dosages as low as 30 microg mL(-1). Hence, MFNs appear to be a valuable and safe bimodal contrast agent that can be developed for the noninvasive diagnosis of breast cancer.

Molecular Imaging of Innate Immune Cell Function in Transplant Rejection

Clinical detection of transplant rejection by repeated endomyocardial biopsy requires catheterization and entails risks. Recently developed molecular and cellular imaging techniques that visualize macrophage host responses could provide a noninvasive alternative. Yet, which macrophage functions may provide useful markers for detecting parenchymal rejection remains uncertain. We transplanted isografts from B6 mice and allografts from Balb/c mice heterotopically into B6 recipients. In this allograft across major histocompatability barriers, the transplanted heart undergoes predictable progressive rejection, leading to graft failure after 1 week. During rejection, crucial macrophage functions, including phagocytosis and release of proteases, render these abundant innate immune cells attractive imaging targets. Two or 6 days after transplantation, we injected either a fluorescent protease sensor or a magnetofluorescent phagocytosis marker. Histological and flow cytometric analyses established that macrophages function as the major cellular signal source. In vivo, we obtained a 3-dimensional functional map of macrophages showing higher phagocytic uptake of magnetofluorescent nanoparticles during rejection using magnetic resonance imaging and higher protease activity in allografts than in isografts using tomographic fluorescence. We further assessed the sensitivity of imaging to detect the degree of rejection. In vivo imaging of macrophage response correlated closely with gradually increasing allograft rejection and attenuated rejection in recipients with a genetically impaired immune response resulting from a deficiency in recombinase-1 (RAG-1(-/-)). Molecular imaging reporters of either phagocytosis or protease activity can detect cardiac allograft rejection noninvasively, promise to enhance the search for novel tolerance-inducing strategies, and have translational potential.

Bio-functionalization of magnetite nanoparticles using an aminophosphonic acid couplingagent: new, ultradispersed, iron-oxide folate nanoconjugates for cancer-specifictargeting

The present study describes a systematic approach towards the design and development ofnovel, bio-functionalized, magneto-fluorescent nanoparticles for cancer-specific targeting.Biocompatible, hydrophilic, magneto-fluorescent nanoparticles with surface-pendant amine,carboxyl or aldehyde groups, to be later used for bio-conjugation, were designed using anaminophosphonic acid coupling agent. These magneto-fluorescent nanoparticles werefurther functionalized with folic acid, using diverse conjugation strategies. A series of newiron-oxide folate nanoconjugates with excellent aqueous dispersion stability and reasonablygood hydrodynamic sizes under a wide range of physiological conditions were developed.These ultradispersed nanosystems were analyzed for their physicochemical properties andcancer-cell targeting ability, facilitated by surface modification with folic acid. Thenanoparticle size, charge, surface chemistry, magnetic properties and colloidal stabilitywere extensively studied using a variety of complementary techniques. Confocalmicroscopy, performed with folate receptor positive human cervical HeLa cancercells, established that these non-cytotoxic iron-oxide folate nanoconjugates wereeffectively internalized by the target cells through receptor-mediated endocytosis.Cell-uptake behaviors of nanoparticles, studied using magnetically activated cell sorting(MACS), clearly demonstrated that cells over-expressing the human folate receptorinternalized a higher level of these nanoparticle–folate conjugates than negative controlcells.

Targeted Nanoparticles for Imaging Incipient Pancreatic Ductal Adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma (PDAC) carries an extremely poor prognosis, typically presenting with metastasis at the time of diagnosis and exhibiting profound resistance to existing therapies. The development of molecular markers and imaging probes for incipient PDAC would enable earlier detection and guide the development of interventive therapies. Here we sought to identify novel molecular markers and to test their potential as targeted imaging agents.Methods and FindingsHere, a phage display approach was used in a mouse model of PDAC to screen for peptides that specifically bind to cell surface antigens on PDAC cells. These screens yielded a motif that distinguishes PDAC cells from normal pancreatic duct cells in vitro, which, upon proteomics analysis, identified plectin-1 as a novel biomarker of PDAC. To assess their utility for in vivo imaging, the plectin-1 targeted peptides (PTP) were conjugated to magnetofluorescent nanoparticles. In conjunction with intravital confocal microscopy and MRI, these nanoparticles enabled detection of small PDAC and precursor lesions in engineered mouse models.ConclusionsOur approach exploited a well-defined model of PDAC, enabling rapid identification and validation of PTP. The developed specific imaging probe, along with the discovery of plectin-1 as a novel biomarker, may have clinical utility in the diagnosis and management of PDAC in humans.

TARGETED NANOPARTICLES FOR IMAGING INCIPIENT PANCREATIC DUCTAL ADENOCARINOMA

Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer death in the United States and shows a rapid clinical course, with a median survival of 6 months and a 5-year survival rate of only 3%. As chemotherapy and radiotherapy have only modest benefits and surgery is only possible in 20% of patients, earliest detection that allows surgical resection offers the best hope for longer survival. The identification of novel molecular markers and the development of imaging probes for pre-neoplastic/early invasive lesions is thus a high priority. We used phage display to identify peptides that distinguish mouse and human PDAC cells from normal pancreatic duct cells in vitro. We subsequently conjugated 2 peptides with the highest affinities and specificities to magnetofluorescent nanoparticles (CLIO-VT680) and demonstrate that these agents can effectively detect emerging tumors and pre-neoplastic lesions in a relevant transgenic mouse model via intravital confocal microscopy (Olympus IV100) and optical/MR imaging (OV-100, Bruker Pharmascan). Correlative histology confirmed the specific tumoral localization of the PDAC targeted agents. Additionally, the peptide-binding partners identified from this approach represent a snapshot of the proteome in aberrant cells and also potential PDAC biomarkers. Using affinity chromatography, we identified the binding partners for several peptides and demonstrate their validity as biomarkers. These specific and sensitive probes may have clinical utility in the diagnosis and management of PDAC in humans.

Multimodality Molecular Imaging Identifies Proteolytic and Osteogenic Activities in Early Aortic Valve Disease

Visualizing early changes in valvular cell functions in vivo may predict the future risk and identify therapeutic targets for prevention of aortic valve stenosis. To test the hypotheses that (1) aortic stenosis shares a similar pathogenesis to atherosclerosis and (2) molecular imaging can detect early changes in aortic valve disease, we used in vivo a panel of near-infrared fluorescence imaging agents to map endothelial cells, macrophages, proteolysis, and osteogenesis in aortic valves of hypercholesterolemic apolipoprotein E-deficient mice (30 weeks old, n=30). Apolipoprotein E-deficient mice with no probe injection (n=10) and wild-type mice (n=10) served as controls. Valves of apolipoprotein E-deficient mice contained macrophages, were thicker than wild-type mice (P<0.001), and showed early dysfunction detected by MRI in vivo. Fluorescence imaging detected uptake of macrophage-targeted magnetofluorescent nanoparticles (24 hours after injection) in apolipoprotein E-deficient valves, which was negligible in controls (P<0.01). Valvular macrophages showed proteolytic activity visualized by protease-activatable near-infrared fluorescence probes. Ex vivo magnetic resonance imaging enhanced with vascular cell adhesion molecule-1-targeted nanoparticles detected endothelial activation in valve commissures, the regions of highest mechanical stress. Osteogenic near-infrared fluorescence signals colocalized with alkaline phosphatase activity and expression of osteopontin, osteocalcin, Runx2/Cbfa1, Osterix, and Notch1 despite no evidence of calcium deposits, which suggests ongoing active processes of osteogenesis in inflamed valves. Notably, the aortic wall contained advanced calcification. Quantitative image analysis correlated near-infrared fluorescence signals with immunoreactive vascular cell adhesion molecule-1, macrophages, and cathepsin-B (P<0.001). Molecular imaging can detect in vivo the key cellular events in early aortic valve disease, including endothelial cell and macrophage activation, proteolytic activity, and osteogenesis.

Labeling of immune cells for in vivo imaging using magnetofluorescent nanoparticles

Observation of immune and stem cells in their native microenvironments requires the development of imaging agents to allow their in vivo tracking. We describe here the synthesis of magnetofluorescent nanoparticles for cell labeling in vitro and for multimodality imaging of administered cells in vivo. MION-47, a prototype monocrystalline iron oxide nanoparticle, was first converted to an intermediate bearing a fluorochrome and amine groups, then reacted with either HIV-Tat peptide or protamine to yield a nanoparticle with membrane-translocating properties. We describe how to assess optimal cell labeling with tests of cell phenotype and function. Synthesis of magnetofluorescent nanoparticles and cell-labeling optimization can be realized in 48 h, whereas nanoparticle uptakes and retention studies may generally take up to 120 h. Labeled cells can be detected by magnetic resonance imaging, fluorescence reflectance imaging, fluorescence-mediated tomography, confocal microscopy and flow cytometry, and can be purified based on their fluorescent or magnetic properties. The present protocol focuses on T-cell labeling but can be used for labeling a variety of circulating cells.

Cellular Imaging of Inflammation in Atherosclerosis Using Magnetofluorescent Nanomaterials

Magnetofluorescent nanoparticles (MFNPs) offer the ability to image cellular inflammation in vivo. To better understand their cellular targeting and imaging capabilities in atherosclerosis, we investigated prototypical dextran-coated near-infrared fluorescent MFNPs in the apolipoprotein E-deficient (apo E-/-) mouse model. In vitro MFNP uptake was highest in activated murine macrophages (p < .001). Apo E-/- mice (n = 11) were next injected with the MFNP (15 mg/kg iron) or saline. In vivo magnetic resonance imaging (MRI) demonstrated strong plaque enhancement by the MFNPs (p < .001 vs. saline), which was confirmed by multimodality ex vivo MRI and fluorescence reflectance imaging. On fluorescence microscopy, MFNPs were found in cellular-rich areas of atheroma and colocalized with immunofluorescent macrophages over endothelial cells and smooth muscle cells (p < .001). Here we show that (1) the in vitro and in vivo cellular distribution of atherosclerosis-targeted MFNPs can be quantified by using fluorescence imaging methods; (2) in atherosclerosis, dextranated MFNPs preferentially target macrophages; and (3) MFNP deposition in murine atheroma can be noninvasively detected by in vivo MRI. This study thus provides a foundation for using MFNPs to image genetic and/or pharmacological perturbations of cellular inflammation in experimental atherosclerosis and for the future development of novel targeted nanomaterials for atherosclerosis.

A Fluorescent Nanosensor for Apoptotic Cells

A biocompatible surface-functionalized nanoparticle was designed to sense phosphatidylserine exposed on apoptotic cells. We conjugated synthetic artificial phosphatidylserine binding ligands in a multivalent fashion onto magnetofluorescent nanoparticles. Our results show that (1) the synthetic nanoparticles bind to apoptotic cells, (2) there is excellent correlation with annexin V staining by microscopy, and (3) FACS analysis with nanoparticles allows the measurement of therapeutic apoptosis induction. The described nanomaterials should be useful for a variety of biomedical applications including in vivo imaging of apoptosis.

An X-ray computed tomography imaging agent based on long-circulating bismuth sulphide nanoparticles

Nanomaterials have become increasingly important in the development of new molecular probes for in vivo imaging, both experimentally and clinically. Nanoparticulate imaging probes have included semiconductor quantum dots, magnetic and magnetofluorescent nanoparticles, gold nanoparticles and nanoshells, among others. However, the use of nanomaterials for one of the most common imaging techniques, computed tomography (CT), has remained unexplored. Current CT contrast agents are based on small iodinated molecules. They are effective in absorbing X-rays, but non-specific distribution and rapid pharmacokinetics have rather limited their microvascular and targeting performance. Here we propose the use of a polymer-coated Bi(2)S(3) nanoparticle preparation as an injectable CT imaging agent. This preparation demonstrates excellent stability at high concentrations (0.25 M Bi(3+)), high X-ray absorption (fivefold better than iodine), very long circulation times (>2 h) in vivo and an efficacy/safety profile comparable to or better than iodinated imaging agents. We show the utility of these polymer-coated Bi(2)S(3) nanoparticles for enhanced in vivo imaging of the vasculature, the liver and lymph nodes in mice. These nanoparticles and their bioconjugates are expected to become an important adjunct to in vivo imaging of molecular targets and pathological conditions.

Development of Nanoparticle Libraries for Biosensing

Magnetic and magnetofluorescent nanoparticles have become important materials for biological applications especially for sensing, separation, and imaging. To achieve target specificity, these nanomaterials are often covalently modified with binding proteins such as antibodies or proteins. Here we report on the creation of nanoparticle libraries that achieve specificity through multivalent modification with small molecules. We explore different synthetic routes to attach small molecules with anhydride, amine, hydroxyl carboxyl, thiol, and epoxy handles. We show that the derived nanomaterials have unique biological functions, possess different behaviors in cell screens, and can be used as substrates for biological screens.

Imaging inflammation of the pancreatic islets in type 1 diabetes.

Type 1 diabetes is the clinical manifestation of aberrant leukocytic infiltration of the pancreatic islets; it is usually diagnosed only very late in disease progression, after the critical autoimmune phenomena have mostly played out. A noninvasive means of directly monitoring the evolution of islet infiltrates would have important research and clinical applications. We have exploited fluorescence and MRI of long-circulating magnetofluorescent nanoparticles to visualize micro-vascular leakage, as an indicator of inflammation, in pancreata of mouse models of type 1 diabetes ex vivo or in vivo. We could detect the onset and evolution of insulitis in vivo and in real time, permitting us to study the natural history of diabetes in individual animals.