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1.
Mol Imaging ; 10(6): 407-19, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22201532

ABSTRACT

Molecular imaging allows clinicians to visualize disease-specific molecules, thereby providing relevant information in the diagnosis and treatment of patients. With advances in genomics and proteomics and underlying mechanisms of disease pathology, the number of targets identified has significantly outpaced the number of developed molecular imaging probes. There has been a concerted effort to bridge this gap with multidisciplinary efforts in chemistry, proteomics, physics, material science, and biology--all essential to progress in molecular imaging probe development. In this review, we discuss target selection, screening techniques, and probe optimization with the aim of developing clinically relevant molecularly targeted imaging agents.


Subject(s)
Molecular Imaging/methods , Molecular Probe Techniques , Molecular Probes/chemistry , Animals , Antibodies/chemistry , DNA/chemistry , Humans , Peptides/chemistry , RNA/chemistry
2.
PLoS One ; 6(7): e22471, 2011.
Article in English | MEDLINE | ID: mdl-21811618

ABSTRACT

The sequencing of the human genome holds out the hope for personalized medicine, but it is clear that analysis of DNA or RNA content alone is not sufficient to understand most disease processes. Proteomic strategies that allow unbiased identification of proteins and their post-transcriptional and -translation modifications are an essential complement to genomic strategies. However, the enormity of the proteome and limitations in proteomic methods make it difficult to determine the targets that are particularly relevant to human disease. Methods are therefore needed that allow rational identification of targets based on function and relevance to disease. Screening methodologies such as phage display, SELEX, and small-molecule combinatorial chemistry have been widely used to discover specific ligands for cells or tissues of interest, such as tumors. Those ligands can be used in turn as affinity probes to identify their cognate molecular targets when they are not known in advance. Here we report an easy, robust and generally applicable approach in which phage particles bearing cell- or tissue-specific peptides serve directly as the affinity probes for their molecular targets. For proof of principle, the method successfully identified molecular binding partners, three of them novel, for 15 peptides specific for pancreatic cancer.


Subject(s)
Biomarkers/metabolism , Proteomics/methods , Amino Acid Sequence , Biomarkers, Tumor/metabolism , Humans , Mass Spectrometry , Molecular Sequence Data , Peptide Library , Peptides/chemistry , Peptides/metabolism , Reproducibility of Results , Tissue Array Analysis
3.
Circ Cardiovasc Imaging ; 2(6): 460-7, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19920044

ABSTRACT

BACKGROUND: A novel dual-contrast molecular MRI technique to image both cardiomyocyte apoptosis and necrosis in vivo within 4 to 6 hours of ischemia is presented. The technique uses the annexin-based nanoparticle AnxCLIO-Cy5.5 (apoptosis) and simultaneous delayed-enhancement imaging with a novel gadolinium chelate, Gd-DTPA-NBD (necrosis). METHODS AND RESULTS: Mice with transient coronary ligation were injected intravenously at the onset of reperfusion with AnxCLIO-Cy5.5 (n=7) or the control probe Inact_CLIO-Cy5.5 (n=6). T2*-weighted MR images (9.4 T) were acquired within 4 to 6 hours of reperfusion. The contrast-to-noise ratio between injured and uninjured myocardium was measured. The mice were then injected with Gd-DTPA-NBD, and delayed-enhancement imaging was performed within 10 to 30 minutes. Uptake of AnxCLIO-Cy5.5 was most prominent in the midmyocardium and was significantly greater than that of Inact_CLIO-Cy5.5 (contrast-to-noise ratio, 8.82+/-1.5 versus 3.78+/-1.1; P<0.05). Only 21+/-3% of the myocardium with accumulation of AnxCLIO-Cy5.5 showed delayed-enhancement of Gd-DTPA-NBD. Wall thickening was significantly reduced in segments with delayed enhancement and/or transmural accumulation of AnxCLIO-Cy5.5 (P<0.001). Fluorescence microscopy of AnxCLIO-Cy5.5 and immunohistochemistry of Gd-DTPA-NBD confirmed the presence of large numbers of apoptotic but potentially viable cardiomyocytes (AnxCLIO-Cy5.5 positive, Gd-DTPA-NBD negative) in the midmyocardium. CONCLUSIONS: A novel technique to image cardiomyocyte apoptosis and necrosis in vivo within 4 to 6 hours of injury is presented and reveals large areas of apoptotic but viable myocardium in the midmyocardium. Strategies to salvage the numerous apoptotic but potentially viable cardiomyocytes in the midmyocardium in acute ischemia should be investigated.


Subject(s)
4-Chloro-7-nitrobenzofurazan/analogs & derivatives , Annexin A5/pharmacokinetics , Annexins/pharmacokinetics , Apoptosis , Carbocyanines/pharmacokinetics , Contrast Media/pharmacokinetics , Gadolinium DTPA/analogs & derivatives , Magnetic Resonance Imaging/methods , Myocardial Infarction/pathology , Myocytes, Cardiac/pathology , Nanoparticles , Necrosis/diagnosis , Organometallic Compounds/pharmacokinetics , Pentetic Acid/analogs & derivatives , 4-Chloro-7-nitrobenzofurazan/pharmacokinetics , Analysis of Variance , Animals , Flow Cytometry , Gadolinium DTPA/pharmacokinetics , Image Processing, Computer-Assisted , In Situ Nick-End Labeling , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , Nanotechnology , Pentetic Acid/pharmacokinetics , Statistics, Nonparametric
4.
Circ Cardiovasc Imaging ; 2(6): 468-75, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19920045

ABSTRACT

BACKGROUND: The ability to image cardiomyocyte (CM) apoptosis in heart failure could facilitate more accurate diagnostics and optimize targeted therapeutics. We thus aimed to develop a platform to image CM apoptosis quantitatively and specifically in heart failure in vivo. The myocardium in heart failure, however, is characterized by very low levels of CM apoptosis and normal vascular permeability, factors thought to preclude the use of molecular MRI. METHODS AND RESULTS: Female mice with overexpression of Gaq were studied. Two weeks postpartum, these mice develop a cardiomyopathy characterized by low levels of CM apoptosis and minimal myocardial necrosis or inflammation. The mice were injected with the annexin-labeled nanoparticle (AnxCLIO-Cy5.5) or a control probe (CLIO-Cy5.5) and imaged in vivo at 9.4 T. Uptake of AnxCLIO-Cy5.5 occurred in isolated clusters, frequently in the subendocardium. Myocardial T2* was significantly lower (7.6+/-1.5 versus 16.8+/-2.7 ms, P<0.05) in the mice injected with AnxCLIO-Cy5.5 versus CLIO-Cy5.5, consistent with the uptake of AnxCLIO-Cy5.5 by apoptotic CMs. A strong correlation (r(2)=0.86, P<0.05) was seen between in vivo T2* (AnxCLIO-Cy5.5 uptake) and myocardial caspase-3 activity. CONCLUSIONS: The ability of molecular MRI to image sparsely expressed targets in the myocardium is demonstrated in this study. Moreover, a novel platform for high-resolution and specific imaging of CM apoptosis in heart failure is established. In addition to providing novel insights into the pathogenesis of CM apoptosis, the developed platform could facilitate the development of novel antiapoptotic therapies in heart failure.


Subject(s)
Annexin A5/pharmacokinetics , Apoptosis , Carbocyanines/pharmacokinetics , Contrast Media/pharmacokinetics , Heart Failure/pathology , Magnetic Resonance Imaging/methods , Myocytes, Cardiac/pathology , Animals , Blotting, Western , Caspase 3/metabolism , Female , Heart Failure/enzymology , Humans , In Situ Nick-End Labeling , Mice , Mice, Transgenic , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/metabolism , Statistics, Nonparametric
5.
Circulation ; 120(7): 592-9, 2009 Aug 18.
Article in English | MEDLINE | ID: mdl-19652086

ABSTRACT

BACKGROUND: Inflammation undermines the stability of atherosclerotic plaques, rendering them susceptible to acute rupture, the cataclysmic event that underlies clinical expression of this disease. Myeloperoxidase is a central inflammatory enzyme secreted by activated macrophages and is involved in multiple stages of plaque destabilization and patient outcome. We report here that a unique functional in vivo magnetic resonance agent can visualize myeloperoxidase activity in atherosclerotic plaques in a rabbit model. METHODS AND RESULTS: We performed magnetic resonance imaging of the thoracic aorta of New Zealand White rabbits fed a cholesterol (n=14) or normal (n=4) diet up to 2 hours after injection of the myeloperoxidase sensor bis-5HT-DTPA(Gd) [MPO(Gd)], the conventional agent DTPA(Gd), or an MPO(Gd) analog, bis-tyr-DTPA(Gd), as controls. Delayed MPO(Gd) images (2 hours after injection) showed focal areas of increased contrast (>2-fold) in diseased wall but not in normal wall (P=0.84) compared with both DTPA(Gd) (n=11; P<0.001) and bis-tyr-DTPA(Gd) (n=3; P<0.05). Biochemical assays confirmed that diseased wall possessed 3-fold elevated myeloperoxidase activity compared with normal wall (P<0.01). Areas detected by MPO(Gd) imaging colocalized and correlated with myeloperoxidase-rich areas infiltrated by macrophages on histopathological evaluations (r=0.91, P<0.0001). Although macrophages were the main source of myeloperoxidase, not all macrophages secreted myeloperoxidase, which suggests that distinct subpopulations contribute differently to atherogenesis and supports our functional approach. CONCLUSIONS: The present study represents a unique approach in the detection of inflammation in atherosclerotic plaques by examining macrophage function and the activity of an effector enzyme to noninvasively provide both anatomic and functional information in vivo.


Subject(s)
Atherosclerosis/enzymology , Atherosclerosis/pathology , Inflammation/enzymology , Inflammation/pathology , Magnetic Resonance Imaging/methods , Peroxidase/metabolism , Animals , Aorta, Thoracic/enzymology , Aorta, Thoracic/pathology , Atherosclerosis/etiology , Disease Models, Animal , Gadolinium DTPA , Hypercholesterolemia/complications , Inflammation/etiology , Macrophages/enzymology , Macrophages/pathology , Male , Rabbits , Regression Analysis
6.
Sens Actuators B Chem ; 138(2): 591-597, 2009 May 06.
Article in English | MEDLINE | ID: mdl-20161242

ABSTRACT

Proteases play important roles in a variety of pathologies from heart disease to cancer. Quantitative measurement of protease activity is possible using a novel spectrally matched dual fluorophore probe and a small animal lifetime imager. The recorded fluorescence from an activatable fluorophore, one that changes its fluorescent amplitude after biological target interaction, is also influenced by other factors including imaging probe delivery and optical tissue absorption of excitation and emission light.Fluorescence from a second spectrally matched constant (non-activatable) fluorophore on each nanoparticle platform can be used to correct for both probe delivery and tissue absorption. The fluorescence from each fluorophore is separated using fluorescence lifetime methods.

8.
Radiology ; 248(3): 925-35, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18647846

ABSTRACT

PURPOSE: To use near-infrared (NIR) optical imaging to assess the therapeutic susceptibility and drug dosing of orthotopic human breast cancers implanted in mice treated with molecularly targeted therapy. MATERIALS AND METHODS: This study was approved by the institutional animal care and use committee. Imaging probes were synthesized by conjugating the human epidermal growth factor receptor type 2 (HER2)-specific antibody trastuzumab with fluorescent dyes. In vitro probe binding was assessed with flow cytometry. HER2-normal and HER2-overexpressing human breast cancer cells were orthotopically implanted in nude mice. Intravital laser scanning fluorescence microscopy was used to evaluate the in vivo association of the probe with the tumor cells. Mice bearing 3-5-mm-diameter tumors were intravenously injected with 0.4 nmol of HER2 probe before or after treatment. A total of 123 mice were used for all in vivo tumor growth and imaging experiments. Tumor fluorescence intensity was assessed, and standard fluorescence values were determined. Statistical significance was determined by performing standard analysis of variance across the imaging cohorts. RESULTS: HER2 probe enabled differentiation between HER2-normal and HER2-overexpressing human breast cancer cells in vitro and in vivo, with binding levels correlating with tumor trastuzumab susceptibility. Serial imaging before and during trastuzumab therapy revealed a significant reduction (P < .05) in probe binding with treatment and thus provided early evidence of successful HER2 inhibition days before the overall reduction in tumor growth was apparent. CONCLUSION: NIR imaging with HER2-specific imaging probes enables evaluation of the therapeutic susceptibility of human mammary tumors and of drug dosing during HER2-targeted therapy with trastuzumab. This approach, combined with tomographic imaging techniques, has potential in the clinical setting for determining patient eligibility for and adequate drug dosing in molecularly targeted cancer therapies.


Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Drug Delivery Systems/methods , Microscopy, Fluorescence/methods , Receptor, ErbB-2/metabolism , Animals , Breast Neoplasms/drug therapy , Cell Line, Tumor , Dose-Response Relationship, Drug , Female , Humans , Mice , Mice, Nude , Molecular Probe Techniques , Prognosis
9.
Bioconjug Chem ; 19(1): 130-7, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17988080

ABSTRACT

Here, we report on NBD-Wm, a fluorescent wortmannin (Wm) probe that maintains the bioactivity of Wm as an inhibitor of PI3 kinase and as an antiproliferative agent. The attachment of the NBD fluorochrome permits NBD-Wm in cells to be monitored by NBD fluorescence-based methods such as FACS or fluorescence microscopy or with an anti-NBD antibody. The fluorescence of NBD-Wm treated cells reached a peak at 1.5 h and then decreased because of the extrusion of a fluorescent compound into the culture media. Cells accumulated NBD-Wm to levels about 30-fold higher than those in the media. NBD-Wm modified five major proteins, with the modification of the catalytic subunit of PI3 kinase being a minor band. The bioactivity of NBD-Wm, coupled with a variety of techniques available for determining its disposition, suggest that NBD-Wm can be a useful tool in understanding the mechanism of action of viridins.


Subject(s)
Androstadienes/metabolism , Androstadienes/pharmacology , Fluorescent Dyes/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Intracellular Space/metabolism , Oxadiazoles/metabolism , Phosphoinositide-3 Kinase Inhibitors , Time Factors , Wortmannin
10.
Mol Imaging Biol ; 8(4): 201-7, 2006.
Article in English | MEDLINE | ID: mdl-16791746

ABSTRACT

PURPOSE: Atherosclerosis is a leading cause of morbidity and mortality in the Western world, yet specific imaging agents to detect and map inflammatory plaques are still lacking. PROCEDURES: We used in vivo phage display to interrogate early atherosclerotic lesions present in ApoE-/- mice with the goal of identifying plaque-associated endothelial cell internalized affinity ligands. RESULTS: We identified 30 phage families with some of these families exhibiting homology to known atherosclerotic proteins, namely, leukemia inhibitory factor, transferrin, and VLA-4. VLA-4 homologous peptides [termed vascular cellular adhesion molecule-1 (VCAM-1) internalizing peptide-28 (VINP28)] bound to and were internalized by VCAM-1-expressing cells and were inhibited by soluble VCAM-1. In addition, a VINP28 modified multimodal nanoparticle showed high affinity for endothelial cells expressing VCAM-1 but low affinity for macrophages or smooth muscle cells. CONCLUSION: The identified peptides represent a set of probes to interrogate the cell surface repertoire and potentially allow early detection of atherosclerosis.


Subject(s)
Affinity Labels , Atherosclerosis/diagnosis , Carotid Stenosis/diagnosis , Peptide Library , Animals , Apolipoproteins E/deficiency , Cells, Cultured , Endothelial Cells , Leukemia Inhibitory Factor Receptor alpha Subunit , Magnetics , Mice , Mice, Mutant Strains , Microscopy, Fluorescence , Myocardium/cytology , Nanostructures , Receptors, Cytokine/metabolism , Receptors, OSM-LIF , Receptors, Transferrin/metabolism , Risk Factors , Sequence Homology, Amino Acid , Vascular Cell Adhesion Molecule-1/metabolism
11.
Neoplasia ; 8(3): 214-22, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16611415

ABSTRACT

Nanoparticles 10 to 100 nm in size can deliver large payloads to molecular targets, but undergo slow diffusion and/or slow transport through delivery barriers. To examine the feasibility of nanoparticles targeting a marker expressed in tumor cells, we used the binding of cyclic arginine-glycine-aspartic acid (RGD) nanoparticle targeting integrins on BT-20 tumor as a model system. The goals of this study were: 1) to use nanoparticles to image alpha(V)beta3 integrins expressed in BT-20 tumor cells by fluorescence-based imaging and magnetic resonance imaging, and, 2) to identify factors associated with the ability of nanoparticles to target tumor cell integrins. Three factors were identified: 1) tumor cell integrin expression (the alpha(V)beta3 integrin was expressed in BT-20 cells, but not in 9L cells); 2) nanoparticle pharmacokinetics (the cyclic RGD peptide cross-linked iron oxide had a blood half-life of 180 minutes and was able to escape from the vasculature over its long circulation time); and 3) tumor vascularization (the tumor had a dense capillary bed, with distances of <100 microm between capillaries). These results suggest that nanoparticles could be targeted to the cell surface markers expressed in tumor cells, at least in the case wherein the nanoparticles and the tumor model have characteristics similar to those of the BT-20 tumor employed here.


Subject(s)
Breast Neoplasms/pathology , Carcinoma/pathology , Fluorometry/methods , Gliosarcoma/pathology , Integrin alphaVbeta3/analysis , Magnetic Resonance Imaging/methods , Nanostructures , Neoplasm Proteins/analysis , Animals , Breast Neoplasms/blood supply , Breast Neoplasms/chemistry , Carcinoma/blood supply , Carcinoma/chemistry , Cell Line, Tumor/chemistry , Cell Line, Tumor/ultrastructure , Diagnostic Imaging/methods , Dose-Response Relationship, Drug , Drug Delivery Systems , Feasibility Studies , Female , Fluorescent Dyes/analysis , Gliosarcoma/chemistry , Half-Life , Humans , Nanostructures/analysis , Neoplasm Transplantation , Oligopeptides/analysis , Particle Size , Rats , Tissue Distribution
12.
Neoplasia ; 7(10): 904-11, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16242073

ABSTRACT

We designed a fluorescent peptide-magnetic nanoparticle conjugate that images E-selectin expression in mouse xenograft models of Lewis lung carcinoma (LLC) by fluorescence reflectance imaging. It was synthesized by attaching the E-selectin-binding peptide (ESBP; CDSDSDITWDQLWDLMK) to a CLIO(Cy5.5) nanoparticle to yield ESBP-CLIO(Cy5.5). Internalization by activated human umbilical vein endothelial cells (HUVECs) was rapid and mediated by E-selectin, indicated by the lack of uptake of nanoparticles bearing similar numbers of a scrambled peptide (Scram). To demonstrate the specificity of E-selectin targeting to ESBP-CLIO(Cy5.5) in vivo, we coinjected ESBP-CLIO(Cy5.5) and Scram-CLIO(Cy3.5) and demonstrated a high Cy5.5/Cy3.5 fluorescence ratio using the LLC. Histology showed that ESBP-CLIO was associated with tumor cells as well as endothelial cells, but fluorescence-activated cell sorter analysis showed a far less expression of E-selectin on LLC than on HUVECs. Using immunohistochemistry, we demonstrated E-selectin expression in both endothelial cells and cancer cells in human prostate cancer specimens. We conclude that ESBP-CLIO(Cy5.5) is a useful probe for imaging E-selectin associated with the LLC tumor, and that E-selectin is expressed not only on endothelial cells but also on LLC cells and human prostate cancer specimens.


Subject(s)
Carcinoma, Lewis Lung/metabolism , E-Selectin/biosynthesis , Nanostructures , Nanotechnology/methods , Animals , Carbocyanines/pharmacology , Cell Line , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Separation , E-Selectin/chemistry , Edetic Acid/chemistry , Endothelial Cells/cytology , Endothelium, Vascular/cytology , Endothelium, Vascular/pathology , Flow Cytometry , Humans , Immunohistochemistry , Interleukin-1/metabolism , Male , Mice , Microscopy, Confocal , Microscopy, Fluorescence , Nanostructures/chemistry , Neoplasm Transplantation , Peptides/chemistry , Platelet Endothelial Cell Adhesion Molecule-1/biosynthesis , Prostatic Neoplasms/metabolism , Sensitivity and Specificity , Substrate Specificity , Time Factors , Umbilical Veins/cytology
13.
Bioconjug Chem ; 16(5): 1240-5, 2005.
Article in English | MEDLINE | ID: mdl-16173804

ABSTRACT

Protamine, a mixture of positively charged proteins from salmon roe used in diverse pharmaceutical applications, was reacted with the N-hydroxysuccinimide ester of tetramethylrhodamine to yield tetramethylrhodamine-labeled protamines (Pro(Rh)) containing one mole of fluorochrome per mole of protein. The internalization of tetramethylrhodamine-labeled protamine (Pro(Rh)) and the fluorescein-labeled tat peptide (Tat(Fl)) showed a similar dependence on time and concentration. Pro(Rh) and Tat(Fl) showed strong nuclear localizations, evident with both live cells and fixed cells co-stained with DAPI, a nuclear stain. The loss of fluorescence when cells were loaded with Pro(Rh) or Tat(Fl) was similar, further supporting a strong similarity between these two materials. Finally, when Pro(Ph) was covalently attached to the amino-CLIO nanoparticle, the cellular uptake of the nanoparticle was greatly enhanced. All experiments were performed with HeLa and CaCo-2 cells with similar results. These observations imply that protamine, a protein in regular clinical use, might be used for the design of novel membrane translocating/nuclear localizing pharmaceuticals whose development was initiated with other membrane-translocating peptides. In addition, the fluorescent protamines developed here might be used to further our understanding of this important pharmaceutical.


Subject(s)
Cell Membrane/metabolism , Protamines/chemistry , Protamines/metabolism , Animals , Caco-2 Cells , Fish Proteins/chemistry , Fish Proteins/metabolism , Fluorescein/chemistry , Gene Products, tat/metabolism , HeLa Cells , Humans , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Transport , Rhodamines/chemistry , Salmon
14.
Magn Reson Med ; 54(3): 718-24, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16086367

ABSTRACT

The ability to image cardiomyocyte apoptosis in vivo with high-resolution MRI could facilitate the development of novel cardioprotective therapies. The sensitivity of the novel nanoparticle AnxCLIO-Cy5.5 for cardiomyocyte apoptosis was thus compared in vitro to that of annexin V-FITC and showed a high degree of colocalization. MRI was then performed, following transient coronary artery (LAD) occlusion, in five mice given AnxCLIO-Cy5.5 and in four mice given an identical dose (2 mg Fe/kg) of CLIO-Cy5.5. MR signal intensity and myocardial T2* were evaluated, in vivo, in hypokinetic regions of myocardium in the LAD distribution. Ex vivo fluorescence imaging was performed to confirm the in vivo findings. Myocardial T2* was significantly lower in the mice given AnxCLIO-Cy5.5 (8.1 versus 13.2 ms, P<0.01), and fluorescence target to background ratio was significantly higher (2.1 versus 1.1, P<0.01). This study thus demonstrates the feasibility of obtaining high-resolution MR images of cardiomyocyte apoptosis in vivo with the novel nanoparticle, AnxCLIO-Cy5.5.


Subject(s)
Annexin A5/pharmacokinetics , Apoptosis , Contrast Media/pharmacokinetics , Magnetic Resonance Imaging/methods , Analysis of Variance , Animals , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , Myocytes, Cardiac , Nanotechnology
15.
Anal Chem ; 77(3): 814-7, 2005 Feb 01.
Article in English | MEDLINE | ID: mdl-15679348

ABSTRACT

Polymer-coated metal or metal oxide nanoparticles have a variety of uses in industry, biological research, and medicine. Characterization of nanoparticles often includes determination of the dimensions of the electron-dense core by transmission electron microscopy (TEM), with the weight of the core determined from core volume and core density. However, TEM is labor intensive, has a long turnaround time, and uses equipment that is sometimes not readily available. Here we present an alternative method for determining the weight of nanoparticle cores termed the viscosity/light scattering method, which uses (i) measurements of viscosity over a wide concentration range to obtain the partial specific volume, (ii) measurements of particle diameter by light scattering, to obtain the volume of an individual particle, and (iii) the concentration of nanoparticles (w/v). We have applied this method to determine the weights of nanoparticle cores (iron of amino-CLIO and ferritin), the weights of globular proteins (molecular weight of IgG and albumin), and the weight of polystyrene microspheres. The viscosity/light scattering method is nondestructive of the sample and can be performed with a variety of materials on a routine basis.


Subject(s)
Nanostructures , Nanotechnology/methods , Weights and Measures , Albumins/chemistry , Amines/chemistry , Ferric Compounds/chemistry , Ferritins/chemistry , Immunoglobulin G/chemistry , Metals/chemistry , Microscopy, Electron, Transmission , Microspheres , Molecular Weight , Oxides/chemistry , Particle Size , Polystyrenes/chemistry , Scattering, Radiation , Viscosity
16.
Chembiochem ; 6(2): 337-45, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15651046

ABSTRACT

We synthesized three peptides, a D-polyarginyl peptide (r8(FITC)), a Tat peptide (Tat(FITC)), and a control peptide (Cp(FITC)) and attached each to amino-CLIO, a nanoparticle 30 nm in diameter. We then examined the effective permeability, Peff, of all six materials through CaCo-2 monolayers. The transport of peptide-nanoparticles was characterized by a lag phase (0-8 h) and a steady-state phase (9-27 h). The steady-state Peff values for peptides were in the order r8(FITC)>Tat(FITC)=Cp(FITC). When r8(FITC) and Tat(FITC) peptides were attached to the nanoparticle, they conferred their propensity to traverse cell monolayers onto the nanoparticle, whereas Cp(FITC) did not. Thus, when the r8(FITC) peptide was attached to the amino-CLIO nanoparticle, the resulting peptide-nanoparticle had a Peff similar to that of this poly-D-arginyl peptide alone. The Peff of r8(FITC)-CLIO (MW approximately 1000 kDa) was similar to that of mannitol (MW=182 Da), a poorly transported reference substance, with a far lower molecular weight. These results are the first to indicate that the modification of nanoparticles by attachment of membrane-translocating sequence-based peptides can alter nanoparticle transport through monolayers. This suggests that the surface modification of nanoparticles might be a general strategy for enhancing the permeability of drugs and that high-permeability nanoparticle-based therapeutics can be useful in selected pharmaceutical applications.


Subject(s)
Biological Transport/physiology , Nanostructures , Peptides/metabolism , Caco-2 Cells , Cells, Cultured , Fluorescein-5-isothiocyanate/chemistry , Fluorescein-5-isothiocyanate/metabolism , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , HeLa Cells , Humans , Molecular Structure , Peptides/chemical synthesis , Peptides/chemistry , Surface Properties
17.
Anal Biochem ; 330(2): 181-5, 2004 Jul 15.
Article in English | MEDLINE | ID: mdl-15203322

ABSTRACT

We have developed a fluorescein isothiocyanate (FITC)-hapten immunoassay, where a FITC-labeled peptide binding to a cell is assayed as the amount of immunoreactive fluorescein present in a cell lysate. An antifluorescein-horseradish peroxidase conjugate binds to either a fluoresceinated peptide in the lysate or a fluorescein attached to the wells of a microtiter plate in a competitive fashion. After washing, solid-phase peroxidase activity is measured and inversely related to the amount of FITC-labeled peptide present. To demonstrate the assay, the interaction of a FITC-labeled bombesin-like peptide with the gastrin-releasing peptide receptor on PC-3 and HT-29 cells was investigated. Using PC-3 cells, we obtained similar displacement curves and numbers of binding sites per cell by both the FITC-hapten immunoassay and a reference radioreceptor assay. The FITC-hapten immunoassay is a sensitive and versatile method, since the same commercially available reagents can be used to assess interactions between any peptide and any receptor. In addition, the FITC-labeled peptide can be used to visualize receptors in fluorescent-activated cell sorting or fluorescent microscopy.


Subject(s)
Cell Communication , Immunoassay/methods , Peptides/analysis , Bombesin , Cell Line , Fluorescein-5-isothiocyanate , Haptens , Horseradish Peroxidase , Humans , Immunoconjugates , Peptides/metabolism , Receptors, Bombesin/metabolism , Receptors, Cell Surface/metabolism
18.
Chembiochem ; 5(3): 275-9, 2004 Mar 05.
Article in English | MEDLINE | ID: mdl-14997519

ABSTRACT

We have developed techniques for the efficient synthesis and screening of small libraries of surface-functionalized nanoparticles for the recognition of specific types of cells. To illustrate this concept we describe the development of a nanoparticle that preferentially recognizes apoptotic Jurkat cells in a manner similar to the apoptosis-recognizing protein annexin V. The nanoparticle, which is detectable by fluorescence or NMR relaxometry, was analyzed for the ability to recognize normal and apoptotic cells by fluorescence-activated cell sorting (FACS) analysis and fluorescence microscopy. The capability to develop nanoparticles which interact with specific target cells could be applied to the design of materials for diverse applications including quantum dots, which serve as fluorescence tracers, colloidal gold, which serves as a tracer for electron micrographs, or the crystalline forms of drugs.


Subject(s)
Apoptosis , Molecular Probes/chemical synthesis , Nanotechnology/methods , Combinatorial Chemistry Techniques , Dextrans , Ferric Compounds , Flow Cytometry , Fluorescence , Humans , Jurkat Cells , Magnetics , Microscopy, Fluorescence , Particle Size , Succinimides
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