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1.  Targeted Nanoparticles for Imaging Incipient Pancreatic Ductal Adenocarcinoma  
PLoS Medicine  2008;5(4):e85.
Background
Pancreatic 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 Findings
Here, 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.
Conclusions
Our 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.
Kimberly Kelly and colleagues describe the discovery of plectin-1 as a novel biomarker for pancreatic ductal adenocarcinoma and the subsequent development of a specific imaging probe using this marker.
Editors' Summary
Background.
Pancreatic cancer is a leading cause of cancer-related death in the US. Like all cancers, it occurs when cells begin to grow uncontrollably and to move around the body (metastasize) because of changes (mutations) in their genes. If pancreatic cancer is found early, surgical removal of the tumor can sometimes provide a cure. Unfortunately, this cancer rarely causes any symptoms in its early stages and the symptoms it does eventually cause—jaundice, abdominal and back pain, and weight loss—are also seen in other illnesses. In addition, even though magnetic resonance imaging (MRI) or other noninvasive imaging techniques can be used to look at the pancreas, by the time tumors are large enough to show up on MRI scans, they have often already spread. Consequently, in most patients, pancreatic cancer is advanced by the time a diagnosis is made, hence surgery is no longer useful. These patients are given radiotherapy and chemotherapy but these treatments are rarely curative and most patients die within a year of diagnosis.
Why Was This Study Done?
If more pancreatic cancers could be found before they had metastasized, it should extend the life expectancy of patients with this type of cancer. An early detection method would be particularly useful for monitoring people at high risk of developing pancreatic cancer. These include people with certain inherited cancer syndromes, pancreatitis (inflammation of the pancreas), and diabetes. Because cancer cells have many mutations, they express different proteins on their cell surface from normal cells. If these proteins could be identified, it might be possible to develop an “imaging probe”—a molecule that binds to a protein found only on cancer cells and that can be detected with MRI, for example—for early detection of pancreatic cancer. In this study, the researchers use a technique called “phage display” to identify several peptides (short sequences of amino acids, the constituent parts of proteins) that specifically bind to pancreatic cancer cells early in their development. They then investigate the possibility of developing an imaging probe from one of these peptides.
What Did the Researchers Do and Find?
The researchers isolated early pancreatic cancer cells from a mouse model of human pancreatic ductal adenocarcinoma (PDAC; the commonest type of pancreatic cancer). Then, by mixing together these cells and normal mouse pancreatic cells with a library of phage clones (phages are viruses that infect bacteria; a clone is a group of genetically identical organisms), each engineered in the laboratory to express a random seven amino-acid peptide, they identified one clone, clone 27, that bound to the mouse tumor cells but not to normal cells. Clone 27 also showed up in the cancer cells in samples of mouse pancreatic intraepithelial neoplasias (PanINs; precursors to pancreatic cancer), mouse PDACs, and human PDACs.
The peptide in clone 27, the researchers report, binds to plectin-1, a protein present both inside and on the membrane of human and mouse PDAC cells but only on the inside of normal pancreatic cells. Finally, the researchers attached this plectin-1–targeted peptide (PTP) to a nanoparticles that was both magnetic and fluorescent (PTP-NP) and used special microscopy (which detects the fluorescent part of this very small particle) and MRI (which detects its magnetic portion) to show that this potential imaging probe was found in areas of PDAC (but not in normal pancreatic tissue) in the mouse model of human PDAC.
What Do These Findings Mean?
These findings identify PTP as a peptide that can distinguish normal pancreatic cells from pancreatic cancer cells. The discovery that plectin-1 (a cytoskeletal component) is abnormally expressed on the cell surface of PDACs provides new information about the development of pancreatic cancer that could eventually lead to new ways to treat this disease. These findings also show that PTP can be used to generate a nanoparticle-based imaging agent that can detect PDAC within a normal pancreas. These results need to be confirmed in people—results obtained in mouse models do not always reflect what happens in people. Nevertheless, they suggest that PTP-NPs might allow the noninvasive detection of early tumors in people at high risk of developing pancreatic cancer, an advance that could extend their lives by identifying tumors earlier, when they can be removed surgically.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050085.
• The Panreatic Cancer Action Network and the Lustgarten Foundation for Pancreatic Cancer Research provide information, support, and advocacy for patients, families, and healthcare professionals
• The MedlinePlus Encyclopedia has a page on pancreatic cancer (in English and Spanish). Links to further information are provided by MedlinePlus
• The US National Cancer Institute has information about pancreatic cancer for patients and health professionals (in English and Spanish)
• The UK charity Cancerbackup also provides information for patients about pancreatic cancer
doi:10.1371/journal.pmed.0050085
PMCID: PMC2292750  PMID: 18416599
2.  Molecular Imaging of Pancreatic Cancer in an Animal Model Using Targeted Multifunctional Nanoparticles 
Gastroenterology  2009;136(5):1514-25.e2.
Background & Aims
Identification of a ligand/receptor system that enables functionalized nanoparticles to efficiently target pancreatic cancer holds great promise for the development of novel approaches for the detection and treatment of pancreatic cancer. Urokinase plasminogen activator receptor (uPAR), a cellular receptor that is highly expressed in pancreatic cancer and tumor stromal cells, is an excellent surface molecule for receptor-targeted imaging of pancreatic cancer using multifunctional nanoparticles.
Methods
The uPAR-targeted dual-modality molecular imaging nanoparticle probe is designed and prepared by conjugating a near-infrared dye-labeled amino-terminal fragment of the receptor binding domain of urokinase plasminogen activator to the surface of functionalized magnetic iron oxide nanoparticles.
Results
We have shown that the systemic delivery of uPAR-targeted nanoparticles leads to their selective accumulation within tumors of orthotopically xenografted human pancreatic cancer in nude mice. The uPAR-targeted nanoparticle probe binds to and is subsequently internalized by uPAR-expressing tumor cells and tumor-associated stromal cells, which facilitates the intratumoral distribution of the nanoparticles and increases the amount and retention of the nanoparticles in a tumor mass. Imaging properties of the nanoparticles enable in vivo optical and magnetic resonance imaging of uPAR-elevated pancreatic cancer lesions.
Conclusions
Targeting uPAR using biodegradable multifunctional nanoparticles allows for the selective delivery of the nanoparticles into primary and metastatic pancreatic cancer lesions. This novel receptor-targeted nanoparticle is a potential molecular imaging agent for the detection of pancreatic cancer.
doi:10.1053/j.gastro.2009.01.006
PMCID: PMC3651919  PMID: 19208341
3.  Mouse lymphatic endothelial cell targeted probes: anti-LYVE-1 antibody-based magnetic nanoparticles 
Purpose
To investigate the specific targeting property of lymphatic vessel endothelial hyaluronan receptor-1 binding polyethylene glycol-coated ultrasmall superparamagnetic iron oxide (LYVE-1-PEG-USPIO) nanoparticles to mouse lymphatic endothelial cells (MLECs).
Methods
A ligand specific target to lymphatic vessels was selected by immunohistochemical staining on the sections of a Lewis subcutaneous transplanted tumor. The z-average hydrodynamic diameter (HD), zeta potential, and the relaxivity of PEG-USPIO and LYVE-1-PEG-USPIO nanoparticles were determined with a laser particle analyzer and magnetic resonance T2 spin echo sequence, respectively. Prussian blue staining and transmission electron microscopy (TEM) of nanoparticle labeled cells were performed to determine the nanoparticles’ binding form. Magnetic resonance imaging (MRI) was performed in vitro to evaluate the signal enhancement on the T2 spin echo sequence of the nanoparticle labeled cells. The iron content of the labeled cells after the Prussian blue staining and MRI scanning was determined by atomic absorption spectroscopy (AAS).
Results
The anti-LYVE-1 antibody was used as the specific ligand to synthesize the target probe to the MLECs. The mean z-average HDs of the LYVE-1-PEG-USPIO and PEG-USPIO nanoparticles were 57.42 ± 0.31 nm and 47.91 ± 0.73 nm, respectively, and the mean zeta potentials of the LYVE-1-PEG-USPIO and PEG-USPIO nanoparticles were 12.38 ± 4.87 mV and 2.57 ± 0.83 m V, respectively. The relaxivities of the LYVE-1-PEG-USPIO and PEG-USPIO nanoparticles were 185.48 mM−1s−1 and 608.32 mM−1s−1. Cells binding nanoparticles were visualized as blue granules in the Prussian blue staining. The TEM results of the labeled cells showed the specific localization of nanoparticles. The AAS results of labeled cells after the Prussian blue staining and MRI scanning showed that the LYVE-1-PEG-USPIO nanoparticles had good binding selectivity for MLECs. MRI results indicated that the PEG-USPIO and LYVE-1-PEG-USPIO nanoparticles could generate contrast on T2-weighted imaging, and the correlation between R2 and the iron content of the labeled cells was significantly positive.
Conclusion
This study demonstrated that LYVE-1-PEG-USPIO nanoparticles might potentially be used as an MRI contrast agent for targeting MLECs, and the magnetic properties of LYVE-1-PEG-USPIO nanoparticles were suitable for MRI.
doi:10.2147/IJN.S45817
PMCID: PMC3693816  PMID: 23818783
nanoparticles; lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1); ultrasmall superparamagnetic iron oxide (USPIO); mouse lymphatic endothelial cells (MLECs); magnetic resonance imaging (MRI)
4.  The future of imaging: developing the tools for monitoring response to therapy in oncology: the 2009 Sir James MacKenzie Davidson Memorial lecture 
The British Journal of Radiology  2010;83(994):814-822.
Since the days of Sir James MacKenzie Davidson, radiology discoveries have been shaping the way patients are managed. The lecture on which this review is based focused not on anatomical imaging, which already has a great impact on patient management, but on the molecular imaging of cancer targets and pathways. In this post-genomic era, we have several tools at our disposal to enable the discovery of new probes for stratifying patients for therapy and for monitoring response to therapy sooner than is possible using conventional cross-sectional imaging methods. I describe a chemical library approach to discovering new imaging agents, as well as novel methods for improving the metabolic stability of existing probes. Finally, I describe the evaluation of these probes for clinical use in both pre-clinical and clinical validation. The chemical library approach is exemplified by the discovery of isatin sulfonamide probes for imaging apoptosis in tumours. This approach allowed important desirable features of radiopharmaceuticals to be incorporated into the design strategy. A lead compound, [18F]ICMT11, is selectively taken up in vitro in cancer cells and in vivo in tumours undergoing apoptosis. Improvement of the metabolic stability of a probe is exemplified by work on [18F]fluoro-[1,2-2H2]choline (“[18F]-D4-choline”), a novel probe for imaging choline metabolism. Deuterium substitution significantly reduced the systemic metabolism of this compound relative to that of non-deuteriated analogues, supporting its future clinical use. In order for probes to be useful for monitoring response a number of validation and/or qualification studies need to be performed, including assessments of whether the probe measures the target or pathway of interest in a specific and reproducible manner, whether the probe is stable to metabolism in vivo, what is the best time to assess response with these probes and finally whether changes in radiotracer uptake are associated with clinical outcome. [18F]Fluorothymidine, a probe for proliferation imaging has been validated and qualified for use in breast cancer. In summary, the ability to create new molecules that can report on specific targets and pathways provides a strategy for studying response to treatment in cancer earlier than it is currently possible. This could fundamentally change the way medicine is practised in the next 5–10 years.
doi:10.1259/bjr/77317821
PMCID: PMC3473744  PMID: 20716650
5.  In Vivo Tumor Cell Targeting with “Click” Nanoparticles 
Bioconjugate chemistry  2008;19(8):1570-1578.
The in vivo fate of nanomaterials strongly determines their biomedical efficacy. Accordingly, much effort has been invested into the development of library screening methods to select targeting ligands for a diversity of sites in vivo. Still, broad application of chemical and biological screens to the in vivo targeting of nanomaterials requires ligand attachment chemistries that are generalizable, efficient, covalent, orthogonal to diverse biochemical libraries, applicable under aqueous conditions, and stable in in vivo environments. To date, the copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition or “click” reaction has shown considerable promise as a method for developing targeted nanomaterials in vitro. Here, we investigate the utility of “click” chemistry for the in vivo targeting of inorganic nanoparticles to tumors. We find that “click” chemistry allows cyclic LyP-1 targeting peptides to be specifically linked to azido-nanoparticles and to direct their binding to p32-expressing tumor cells in vitro. Moreover, “click” nanoparticles are able to stably circulate for hours in vivo following intravenous administration (>5h circulation time), extravasate into tumors, and penetrate the tumor interstitium to specifically bind p32-expressing cells in tumors. In the future, in vivo use of “click” nanomaterials should expedite the progression from ligand discovery to in vivo evaluation and diversify approaches toward multifunctional nanoparticle development.
doi:10.1021/bc800077y
PMCID: PMC2538627  PMID: 18611045
6.  MRl of Prostate Cancer Antigen Expression for Diagnosis and lmmunotherapy 
PLoS ONE  2012;7(6):e38350.
Background
Tumor antigen (TA)–targeted monoclonal antibody (mAb) immunotherapy can be effective for the treatment of a broad range of cancer etiologies; however, these approaches have demonstrated variable clinical efficacy for the treatment of patients with prostate cancer (PCa). An obstacle currently impeding translational progress has been the inability to quantify the mAb dose that reaches the tumor site and binds to the targeted TAs. The coupling of mAb to nanoparticle-based magnetic resonance imaging (MRI) probes should permit in vivo measurement of patient-specific biodistributions; these measurements could facilitate future development of novel dosimetry paradigms wherein mAb dose is titrated to optimize outcomes for individual patients.
Methods
The prostate stem cell antigen (PSCA) is broadly expressed on the surface of prostate cancer (PCa) cells. Anti-human PSCA monoclonal antibodies (mAb 7F5) were bound to Au/Fe3O4 (GoldMag) nanoparticles (mAb 7F5@GoldMag) to serve as PSCA-specific theragnostic MRI probe permitting visualization of mAb biodistribution in vivo. First, the antibody immobilization efficiency of the GoldMag particles and the efficacy for PSCA-specific binding was assessed. Next, PC-3 (prostate cancer with PSCA over-expression) and SMMC-7721 (hepatoma cells without PSCA expression) tumor-bearing mice were injected with mAb 7F5@GoldMag for MRI. MRI probe biodistributions were assessed at increasing time intervals post-infusion; therapy response was evaluated with serial tumor volume measurements.
Results
Targeted binding of the mAb 7F5@GoldMag probes to PC-3 cells was verified using optical images and MRI; selective binding was not observed for SMMC-7721 tumors. The immunotherapeutic efficacy of the mAb 7F5@GoldMag in PC-3 tumor-bearing mice was verified with significant inhibition of tumor growth compared to untreated control animals.
Conclusion
Our promising results suggest the feasibility of using mAb 7F5@GoldMag probes as a novel paradigm for the detection and immunotherapeutic treatment of PCa. We optimistically anticipate that the approaches have the potential to be translated into the clinical settings.
doi:10.1371/journal.pone.0038350
PMCID: PMC3384648  PMID: 22761679
7.  Combinatorial Ligand-directed Lung Targeting 
Phage display of random peptide libraries is a powerful, unbiased method frequently used to discover ligands for virtually any protein of interest and to reveal functional protein–protein interaction partners. Moreover, in vivo phage display permits selection of peptides that bind specifically to different vascular beds without any previous knowledge pertaining to the nature of their corresponding receptors. Vascular targeting exploits molecular differences inherent in blood vessels within given organs and tissues, as well as diversity between normal and angiogenic blood vessels. Over the years, our group has identified phage capable of homing to lung blood vessels based on screenings using immortalized lung endothelial cells combined with in vivo selections after intravenous administration of combinatorial libraries. Peptides targeting lung vasculature have been extensively characterized and a lead homing peptide has shown interesting biological properties, bringing novel insights as to the implications of lung endothelial cell apoptosis in the pathogenesis of emphysema. We have also designed and developed targeted nanoparticles with imaging capabilities and/or drug delivery functions by combining phage display technology and elemental gold (Au) nanoparticles, constituting a promising platform for the development of therapeutic agents for imaging and treatment of lung disorders. Given the important role of the endothelium in the pathogenesis and progression of several diseases associated with the airways, ligand-directed discovery of lung vascular markers is an important milestone toward the development of future targeted therapies.
doi:10.1513/pats.200903-014AW
PMCID: PMC3266014  PMID: 19687212
vascular targeting; lung; phage display
8.  Bioconjugation of Ultrabright Semiconducting Polymer Dots for Specific Cellular Targeting 
Journal of the American Chemical Society  2010;132(43):15410-15417.
Semiconducting polymer dots (Pdots) represent a new class of ultrabright fluorescent probes for biological imaging. They exhibit several important characteristics for experimentally demanding in vitro and in vivo fluorescence studies, such as their high brightness, fast emission rate, excellent photostability, non-blinking, and non-toxic feature. However, controlling the surface chemistry and bioconjugation of Pdots has been a challenging problem that prevented their widespread applications in biological studies. Here, we report a facile yet powerful conjugation method that overcomes this challenge. Our strategy for Pdot functionalization is based on entrapping heterogeneous polymer chains into a single dot, driven by hydrophobic interactions during nanoparticle formation. A small amount of amphiphilic polymer bearing functional groups is co-condensed with the majority of semiconducting polymers to modify and functionalize the nanoparticle surface for subsequent covalent conjugation to biomolecules, such as streptavidin and immunoglobulin G (IgG). The Pdot bioconjugates can effectively and specifically label cellular targets, such as cell surface marker in human breast cancer cells, without any detectable non-specific binding. Single-particle imaging, cellular imaging, and flow cytometry experiments indicate a much higher fluorescence brightness of Pdots compared to those of Alexa dye and quantum dot probes. The successful bioconjugation of these ultrabright nanoparticles presents a novel opportunity to apply versatile semiconducting polymers to various fluorescence measurements in modern biology and biomedicine.
doi:10.1021/ja107196s
PMCID: PMC2965818  PMID: 20929226
9.  Imaging mRNA expression levels in living cells with PNA·DNA binary FRET probes delivered by cationic shell-crosslinked nanoparticles 
Organic & biomolecular chemistry  2013;11(19):3159-3167.
Optical imaging of gene expression through the use of fluorescent antisense probes targeted to the mRNA has been an area of great interest. The main obstacles to developing highly sensitive antisense fluorescent imaging agents has been the inefficient intracellular delivery of the probes and high background signal from unbound probes. Binary antisense probes have shown great promise as mRNA imaging agents because a signal can only occur if both probes are bound simultaneously to the mRNA target site. Selecting an accessible binding site is made difficult by RNA folding and protein binding in vivo and the need to bind two probes. Even more problematic, has been a lack of methods for efficient cytoplasmic delivery of the probes that would be suitable for eventual applications in vivo applications in animals. Herein we report the imaging of iNOS mRNA expression in live mouse macrophage cells with PNA·DNA binary FRET probes delivered by a cationic shell crosslinked knedel-like nanoparticle (cSCK). We first demonstrate that FRET can be observed on in vitro transcribed mRNA with both the PNA probes and the PNA•DNA hybrid probes. We then demonstrate that the FRET signal can be observed in live cells when the hybrid probes are transfected with the cSCK, and that the strength of the FRET signal is sequence specific and depends on the mRNA expression level.
doi:10.1039/c3ob26923j
PMCID: PMC3687806  PMID: 23538604
10.  M13-templated magnetic nanoparticles for targeted in vivo imaging of prostate cancer 
Nature nanotechnology  2012;7(10):677-682.
Molecular imaging allows clinicians to visualize the progression of tumours and obtain relevant information for patient diagnosis and treatment1. Owing to their intrinsic optical, electrical and magnetic properties, nanoparticles are promising contrast agents for imaging dynamic molecular and cellular processes such as protein-protein interactions, enzyme activity or gene expression2. Until now, nanoparticles have been engineered with targeting ligands such as antibodies and peptides to improve tumour specificity and uptake. However, excessive loading of ligands can reduce the targeting capabilities of the ligand3,4,5 and reduce the ability of the nanoparticle to bind to a finite number of receptors on cells6. Increasing the number of nanoparticles delivered to cells by each targeting molecule would lead to higher signal-to-noise ratios and improve image contrast. Here, we show that M13 filamentous bacteriophage can be used as a scaffold to display targeting ligands and multiple nanoparticles for magnetic resonance imaging of cancer cells and tumours in mice. Monodisperse iron oxide magnetic nanoparticles assemble along the M13 coat, and its distal end is engineered to display a peptide that targets SPARC glycoprotein, which is overexpressed in various cancers. Compared with nanoparticles that are directly functionalized with targeting peptides, our approach improves contrast because each SPARC-targeting molecule delivers a large number of nanoparticles into the cells. Moreover, the targeting ligand and nanoparticles could be easily exchanged for others, making this platform attractive for in vivo high-throughput screening and molecular detection.
doi:10.1038/nnano.2012.146
PMCID: PMC4059198  PMID: 22983492
11.  Tumor angiogenic endothelial cell targeting by a novel integrin-targeted nanoparticle 
Angiogenesis is an important process in cancer growth and metastasis. During the tumor angiogenic process, endothelial cells express various cell surface receptors which can be utilized for molecular imaging and targeted drug delivery. One such protein receptor of interest is the integrin αvβ3. Our group is involved in the development of molecular imaging probes and drug delivery systems targeting αvβ3. Based on extensive lead optimization study with the integrin antagonist compounds, we have developed a new generation of integrin αvβ3 compound (IA) which has superior binding affinity to αvβ3. Utilizing this IA as a targeting agent, we have developed a novel integrin-targeted nanoparticle (ITNP) system for targeted drug delivery and imaging of cancer angiogenesis. When multiple copies of the IA were conjugated onto the nanoparticle surface, strong and selective binding to the integrin αvβ3 was observed. These ITNPs also were rapidly taken up by cells that express αvβ3. The ITNPs accumulated in the angiogenic vessels, after systemic administration in a murine squamous cell carcinoma model. This novel intergrin targeted ITNP platform will likely have an application in targeted delivery of drugs and genes in vivo and can also be used for molecular imaging.
PMCID: PMC2676654  PMID: 18019845
targeted nanoparticle; integrin αvβ3; cancer; angiogenesis
12.  Synthesis and application of luminescent single CdS quantum dot encapsulated silica nanoparticles directed for precision optical bioimaging 
This paper presents the synthesis of aqueous cadmium sulfide (CdS) quantum dots (QDs) and silica-encapsulated CdS QDs by reverse microemulsion method and utilized as targeted bio-optical probes. We report the role of CdS as an efficient cell tag with fluorescence on par with previously documented cadmium telluride and cadmium selenide QDs, which have been considered to impart high levels of toxicity. In this study, the toxicity of bare QDs was efficiently quenched by encapsulating them in a biocompatible coat of silica. The toxicity profile and uptake of bare CdS QDs and silica-coated QDs, along with the CD31-labeled, silica-coated CdS QDs on human umbilical vein endothelial cells and glioma cells, were investigated. The effect of size, along with the time-dependent cellular uptake of the nanomaterials, has also been emphasized. Enhanced, high-specificity imaging toward endothelial cell lines in comparison with glioma cells was achieved with CD31 antibody-conjugated nanoparticles. The silica-coated nanomaterials exhibited excellent biocompatibility and greater photostability inside live cells, in addition to possessing an extended shelf life. In vivo biocompatibility and localization study of silica-coated CdS QDs in medaka fish embryos, following direct nanoparticle exposure for 24 hours, authenticated the nanomaterials’ high potential for in vivo imaging, augmented with superior biocompatibility. As expected, CdS QD-treated embryos showed 100% mortality, whereas the silica-coated QD-treated embryos stayed viable and healthy throughout and after the experiments, devoid of any deformities. We provide highly cogent and convincing evidence for such silica-coated QDs as a model nanoparticle in practice, to achieve in vitro and in vivo precision targeted imaging.
doi:10.2147/IJN.S31310
PMCID: PMC3414225  PMID: 22888233
endothelial imaging; CD31; silica nanoparticle; CdS QDs; medaka embryos; biocompatibility
13.  Endothelial Targeting of Cowpea Mosaic Virus (CPMV) via Surface Vimentin 
PLoS Pathogens  2009;5(5):e1000417.
Cowpea mosaic virus (CPMV) is a plant comovirus in the picornavirus superfamily, and is used for a wide variety of biomedical and material science applications. Although its replication is restricted to plants, CPMV binds to and enters mammalian cells, including endothelial cells and particularly tumor neovascular endothelium in vivo. This natural capacity has lead to the use of CPMV as a sensor for intravital imaging of vascular development. Binding of CPMV to endothelial cells occurs via interaction with a 54 kD cell-surface protein, but this protein has not previously been identified. Here we identify the CPMV binding protein as a cell-surface form of the intermediate filament vimentin. The CPMV-vimentin interaction was established using proteomic screens and confirmed by direct interaction of CPMV with purified vimentin, as well as inhibition in a vimentin-knockout cell line. Vimentin and CPMV were also co-localized in vascular endothelium of mouse and rat in vivo. Together these studies indicate that surface vimentin mediates binding and may lead to internalization of CPMV in vivo, establishing surface vimentin as an important vascular endothelial ligand for nanoparticle targeting to tumors. These results also establish vimentin as a ligand for picornaviruses in both the plant and animal kingdoms of life. Since bacterial pathogens and several other classes of viruses also bind to surface vimentin, these studies suggest a common role for surface vimentin in pathogen transmission.
Author Summary
Cowpea mosaic virus (CPMV), a plant virus that does not replicate in animals, is extensively used in material science and nanobiotechnology. CPMV has been found to specifically interact with mammalian cells after oral or intravenous administration, as well as in intravital vascular imaging studies that used a fluorescently modified form of CPMV. Binding of CPMV to mammalian cells was shown to be via a cell-surface binding protein (CPMV-BP). Herein we identify this cell surface CPMV-BP through biochemical analysis, live cell experiments, and animal models. We found this surface exposed protein to be vimentin. Vimentin is principally a cytoskeletal protein that functions in the interior of cells to modulate architecture and dynamics. Our results now indicate surface vimentin can be used as a vascular endothelial marker and targeting option on the exterior surface of these cells. This work also unifies the relationship between CPMV and closely related mammalian viruses such as poliovirus, Theiler's murine encephalomyelitis virus (TMEV), and coxsackie virus through the collective use of vimentin during their infectious cycle. Several other bacterial and viral pathogens use surface vimentin as an attachment receptor as well, and this research may lead to the development of broad-spectrum strategies to inhibit infection.
doi:10.1371/journal.ppat.1000417
PMCID: PMC2670497  PMID: 19412526
14.  Spectral Analysis of Multiplex Raman Probe Signatures 
ACS nano  2008;2(11):2306-2314.
Raman nanoparticle probes are an emerging new class of optical labels for interrogation of physiological and pathological processes in bioassays, cells, and tissues. Although their unique emission signatures are ideal for multiplexing, the full potential of these probes has not been realized because conventional analysis methods are inadequate. We report a novel spectral fitting method that exploits the entire spectral signature to quantitatively extract individual probe signals from multiplex spectra. We evaluate the method in a series of multiplex assays using unconjugated and antibody-conjugated Composite Organic-Inorganic Nanoparticles (COINs). Results show sensitive multiplex detection of small signals (<2% of total signal), and similar detection limits in corresponding 4-plex and singlet plate binding assays. In a triplex assay on formalin-fixed human prostate tissue, two antibody-conjugated COINs and a conventional fluorophore are used to image expression of prostate-specific antigen, cytokeratin-18, and DNA. The spectral analysis method effectively removes tissue autofluorescence and other unknown background, allowing accurate and reproducible imaging (area under ROC curve 0.89+/−0.03) at subcellular spatial resolution. In all assay systems, the error attributable to spectral analysis constitutes ≤2% of total signal. In summary, the spectral fitting method provides (1) quantification of signals from multiplex spectra with overlapping peaks, (2) robust spot-by-spot removal of unknown background, (3) the opportunity to quantitatively assess the analysis error, (4) elimination of operator bias, and (5) simple automation appropriate for high-throughput analysis. The simple implementation and universal applicability of this approach significantly expands the potential of Raman probes for quantitative in-vivo and ex-vivo multiplex analysis.
doi:10.1021/nn800243g
PMCID: PMC2662378  PMID: 19206397
Composite Organic-Inorganic Nanoparticles (COINs); surface-enhanced Raman scattering (SERS); multiplex assays; tissue imaging; prostate-specific antigen; cytokeratin-18
15.  Magnetomotive Molecular Nanoprobes 
Current Medicinal Chemistry  2011;18(14):2103-2114.
Tremendous developments in the field of biomedical imaging in the past two decades have resulted in the transformation of anatomical imaging to molecular-specific imaging. The main approaches towards imaging at a molecular level are the development of high resolution imaging modalities with high penetration depths and increased sensitivity, and the development of molecular probes with high specificity. The development of novel molecular contrast agents and their success in molecular optical imaging modalities have lead to the emergence of molecular optical imaging as a more versatile and capable technique for providing morphological, spatial, and functional information at the molecular level with high sensitivity and precision, compared to other imaging modalities. In this review, we discuss a new class of dynamic contrast agents called magnetomotive molecular nanoprobes for molecular-specific imaging. Magnetomotive agents are superparamagnetic nanoparticles, typically iron-oxide, that are physically displaced by the application of a small modulating external magnetic field. Dynamic phase-sensitive position measurements are performed using any high resolution imaging modality, including optical coherence tomography (OCT), ultrasonography, or magnetic resonance imaging (MRI). The dynamics of the magnetomotive agents can be used to extract the biomechanical tissue properties in which the nanoparticles are bound, and the agents can be used to deliver therapy via magnetomotive displacements to modulate or disrupt cell function, or hyperthermia to kill cells. These agents can be targeted via conjugation to antibodies, and in vivo targeted imaging has been shown in a carcinogen-induced rat mammary tumor model. The iron-oxide nanoparticles also exhibit negative T2 contrast in MRI, and modulations can produce ultrasound imaging contrast for multimodal imaging applications.
PMCID: PMC3319747  PMID: 21517766
Magnetomotion; molecular imaging; optical coherence tomography; hyperthermia; superparamagnetic iron oxide nanoparticles; targeting
16.  Detection of Hydroxyapatite in Calcified Cardiovascular Tissues 
Atherosclerosis  2012;224(2):340-347.
Objective
The objective of this study is to develop a method for selective detection of the calcific (hydroxyapatite) component in human aortic smooth muscle cells in vitro and in calcified cardiovascular tissues ex vivo. This method uses a novel optical molecular imaging contrast dye, Cy-HABP-19, to target calcified cells and tissues.
Methods
A peptide that mimics the binding affinity of osteocalcin was used to label hydroxyapatite in vitro and ex vivo. Morphological changes in vascular smooth muscle cells were evaluated at an early stage of the mineralization process induced by extrinsic stimuli, osteogenic factors and a magnetic suspension cell culture. Hydroxyapatite components were detected in monolayers of these cells in the presence of osteogenic factors and a magnetic suspension environment.
Results
Atherosclerotic plaque contains multiple components including lipidic, fibrotic, thrombotic, and calcific materials. Using optical imaging and the Cy-HABP-19 molecular imaging probe, we demonstrated that hydroxyapatite components could be selectively distinguished from various calcium salts in human aortic smooth muscle cells in vitro and in calcified cardiovascular tissues, carotid endarterectomy samples and aortic valves, ex vivo.
Conclusion
Hydroxyapatite deposits in cardiovascular tissues were selectively detected in the early stage of the calcification process using our Cy-HABP-19 probe. This new probe makes it possible to study the earliest events associated with vascular hydroxyapatite deposition at the cellular and molecular levels. This target-selective molecular imaging probe approach holds high potential for revealing early pathophysiological changes, leading to progression, regression, or stabilization of cardiovascular diseases.
doi:10.1016/j.atherosclerosis.2012.07.023
PMCID: PMC3459140  PMID: 22877867
atherosclerosis; calcification; hydroxyapatite; molecular imaging; osteocalcin
17.  In Vitro and In Vivo Platelet Targeting By Cyclic RGD-modified Liposomes 
Cell-selective delivery using ligand-decorated nanoparticles is a promising modality for treating cancer and vascular diseases. We are developing liposome nanoparticles surface-modified by RGD peptide ligands having targeting specificity to integrin GPIIb-IIIa. This integrin is upregulated and stimulated into a ligand-binding conformation on the surface activated platelets. Activated platelet adhesion and aggregation are primary events in atherosclerosois, thrombosis and restenosis. Hence platelet-targeted nanoparticles hold the promise of vascular site-selective delivery of drugs and imaging probes. Here, we report in vitro and ex vivo microscopy studies of platelet-targeting by liposomes surface-modified with a cyclic RGD peptide. The peptide-modified liposomes were labeled either with a lipophilic fluorophore or with lipid-tethered Nanogold®. For in vitro tests, coverslip-adhered activated human platelets were incubated with probe-labeled liposomes, followed by analysis with fluorescence and phase contrast microscopy, and scanning electron microscopy (SEM). For in vivo tests, the liposomes were introduced within a catheter-injured carotid artery restenosis model in rats and post-euthanasia, the artery was imaged ex vivo by fluorescence microscopy and SEM. All microscopy results showed successful platelet-targeting by the peptide-modified liposomes. The in vitro SEM results also enabled visualization of nanoscopic liposomes attached to activated platelets. The results validate our nanoparticle design for site-selective vascular delivery.
doi:10.1002/jbm.a.32549
PMCID: PMC2854838  PMID: 19743511
platelets; targeting; drug delivery; cyclic RGD
18.  Discovery of Selective Probes and Antagonists for G Protein-Coupled Receptors FPR/FPRL1 and GPR30 
Recent technological advances in flow cytometry provide a versatile platform for high throughput screening of compound libraries coupled with high-content biological testing and drug discovery. The G protein-coupled receptors (GPCRs) constitute the largest class of signaling molecules in the human genome with frequent roles in disease pathogenesis, yet many examples of orphan receptors with unknown ligands remain. The complex biology and potential for drug discovery within this class provide strong incentives for chemical biology approaches seeking to develop small molecule probes to facilitate elucidation of mechanistic pathways and enable specific manipulation of the activity of individual receptors. We have initiated small molecule probe development projects targeting two distinct families of GPCRs: the formylpeptide receptors (FPR/FPRL1) and G protein-coupled estrogen receptor (GPR30). In each case the assay for compound screening involved the development of an appropriate small molecule fluorescent probe, and the flow cytometry platform provided inherently biological rich assays that enhanced the process of identification and optimization of novel antagonists. The contributions of cheminformatics analysis tools, virtual screening, and synthetic chemistry in synergy with the biomolecular screening program have yielded valuable new chemical probes with high binding affinity, selectivity for the targeted receptor, and potent antagonist activity. This review describes the discovery of novel small molecule antagonists of FPR and FPRL1, and GPR30, and the associated characterization process involving secondary assays, cell based and in vivo studies to define the selectivity and activity of the resulting chemical probes
PMCID: PMC2885834  PMID: 19807662
flow cytometry; fluorescent; GPCR; formylpeptide receptor; inflammation; GPR30; GPER; estrogen; nongenomic; cancer; antidepressant
19.  Molecular profiling of single cancer cells and clinical tissue specimens with semiconductor quantum dots 
Semiconductor quantum dots (QDs) are a new class of fluorescent labels with broad applications in biomedical imaging, disease diagnostics, and molecular and cell biology. In comparison with organic dyes and fluorescent proteins, quantum dots have unique optical and electronic properties such as size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. Recent advances have led to multifunctional nanoparticle probes that are highly bright and stable under complex in vitro and in vivo conditions. New designs involve encapsulating luminescent QDs with amphiphilic block copolymers, and linking the polymer coating to tumor-targeting ligands and drug-delivery functionalities. These improved QDs have opened new possibilities for real-time imaging and tracking of molecular targets in living cells, for multiplexed analysis of biomolecular markers in clinical tissue specimens, and for ultrasensitive imaging of malignant tumors in living animal models. In this article, we briefly discuss recent developments in bioaffinity QD probes and their applications in molecular profiling of individual cancer cells and clinical tissue specimens.
PMCID: PMC2676641  PMID: 17722280
nanoparticle; nanocrystal; semiconductor; cancer; tumor; tissue section; live cell
20.  Multifunctional nanoagent for thrombus-targeted fibrinolytic therapy 
Nanomedicine (London, England)  2012;7(7):1017-1028.
Aims
Current thrombolytic therapies rely upon exogenous plasminogen activators (PA) to effectively lyse clots, thereby restoring blood flow and preventing tissue and organ death. Yet, these PAs may also impair normal hemostasis which may lead to life-threatening bleeding, including intracerebral hemorrhage. Thus, the aim of this current study is to develop new thrombus-targeted fibrinolytic agents that harness the multifunctional theranostic capabilities of nanomaterials, potentially allowing for the generation of efficacious thrombolytics while minimizing deleterious side effects.
Materials and Methods
A thrombus-targeted nano-fibrinolytic agent (CLIO-FXIII-PEG-tPA) was synthesized using a magnetofluorescent crosslinked dextran-coated iron oxide (CLIO) nanoparticle platform that was conjugated to recombinant tissue plasminogen activator (tPA). Thrombus-targeting was achieved by derivatizing the nanoparticle with an activated factor XIII (FXIIIa)-sensitive peptide based on the amino terminus of α2-antiplasmin. Human plasma clot binding ability of the targeted and control agents was assessed by fluorescence reflectance imaging. Next, the in vitro enzymatic activity of the agents was assessed by S2288-based amidolytic activity, and an ELISA D-dimer assay for fibrinolysis. In vivo targeting of the nanoagent was next examined by intravital fluorescence microscopy of murine arterial and venous thrombosis. The fibrinolytic activity of the targeted nanoagent compared to free tPA was then evaluated in vivo in murine pulmonary embolism.
Results
In vitro, the targeted thrombolytic nanoagent demonstrated binding to fresh frozen plasma (FFP) clots superior to control nanoagents (ANOVA p < 0.05). On a weight (mg) basis, the S2288 amidolytic efficiency of the targeted nanoagent was approximately 15% reduced compared to free tPA. When normalized by S2288-based activity, targeted, control, and free tPA samples demonstrated equivalent in vitro fibrinolytic activity against human plasma clots, as determined by ELISA D-dimer assays. The FXIIIa targeted fibrinolytic nanoagent efficiently bound the margin of intravascular thrombi as detected by IVFM. In in vivo fibrinolysis studies normalized for activity, the FXIIIa-targeted agent lysed pulmonary emboli with similar efficacy as free tPA (p>0.05).
Conclusions
The applicability of a FXIIIa-targeted thrombolytic nanoagent in the treatment of thromboembolism was demonstrated in vitro and in vivo. Future studies are planned to investigate the safety profile and overall efficacy of this class of nanoagents, and to further optimize their thrombus-targeting profile and lytic action.
doi:10.2217/nnm.11.179
PMCID: PMC3360120  PMID: 22348271
Fibrinolytic; iron oxide; therapy; thrombosis; multimodal; theranostic; imaging
21.  High Resolution Fluorescence Imaging of Cancers Using Lanthanide Ion-Doped Upconverting Nanocrystals 
Cancers  2012;4(4):1067-1105.
During the last decade inorganic luminescent nanoparticles that emit visible light under near infrared (NIR) excitation (in the biological window) have played a relevant role for high resolution imaging of cancer. Indeed, semiconductor quantum dots (QDs) and metal nanoparticles, mostly gold nanorods (GNRs), are already commercially available for this purpose. In this work we review the role which is being played by a relatively new class of nanoparticles, based on lanthanide ion doped nanocrystals, to target and image cancer cells using upconversion fluorescence microscopy. These nanoparticles are insulating nanocrystals that are usually doped with small percentages of two different rare earth (lanthanide) ions: The excited donor ions (usually Yb3+ ion) that absorb the NIR excitation and the acceptor ions (usually Er3+, Ho3+ or Tm3+), that are responsible for the emitted visible (or also near infrared) radiation. The higher conversion efficiency of these nanoparticles in respect to those based on QDs and GNRs, as well as the almost independent excitation/emission properties from the particle size, make them particularly promising for fluorescence imaging. The different approaches of these novel nanoparticles devoted to “in vitro” and “in vivo” cancer imaging, selective targeting and treatment are examined in this review.
doi:10.3390/cancers4041067
PMCID: PMC3712733  PMID: 24213500
cancer; fluorescence imaging; lanthanide-doped nanocrystals; upconversion; targeting
22.  Combined Small Interfering RNA Therapy and In Vivo Magnetic Resonance Imaging in Islet Transplantation 
Diabetes  2011;60(2):565-571.
OBJECTIVE
Recent advances in human islet transplantation are hampered by significant graft loss shortly after transplantation and inability to follow islet fate directly. Both issues were addressed by utilizing a dual-purpose therapy/imaging small interfering RNA (siRNA)-nanoparticle probe targeting apoptotic-related gene caspase-3. We expect that treatment with the probe would result in significantly better survival of transplanted islets, which could be monitored by in vivo magnetic resonance imaging (MRI).
RESEARCH DESIGN AND METHODS
We synthesized a probe consisting of therapeutic (siRNA to human caspase-3) and imaging (magnetic iron oxide nanoparticles, MN) moieties. In vitro testing of the probe included serum starvation of the islets followed by treatment with the probe. Caspase-3 gene silencing and protein expression were determined by RT-PCR and Western blot, respectively. In vivo studies included serial MRI of NOD-SCID mice transplanted with MN-small interfering (si)Caspase-3–labeled human islets under the left kidney capsule and MN-treated islets under the right kidney capsule.
RESULTS
Treatment with MN-siCaspase-3 probe resulted in decrease of mRNA and protein expression in serum-starved islets compared with controls. In vivo MRI showed that there were significant differences in the relative volume change between MN-siCaspase-3–treated grafts and MN-labeled grafts. Histology revealed decreased caspase-3 expression and cell apoptosis in MN-siCaspase-3–treated grafts compared with the control side.
CONCLUSIONS
Our data show the feasibility of combining siRNA therapy and in vivo monitoring of transplanted islets in mice. We observed a protective effect of MN-siCaspase-3 in treated islets both in vitro and in vivo. This study could potentially aid in increasing the success of clinical islet transplantation.
doi:10.2337/db10-1400
PMCID: PMC3028356  PMID: 21270267
23.  Peptide mediated cancer targeting of nanoconjugates 
Targeted use of nanoparticles in vitro, in cells and in vivo requires nanoparticle surface functionalization. Moieties that can be used for such a purpose include small molecules as well as polymers made of different biological and organic materials. Short amino acid polymers--peptides can often rival target binding avidity of much larger molecules. At the same time, peptides are smaller than most nanoparticles and thus allow for multiple nanoparticle modifications and creation of pluripotent nanoparticles. Most nanoparticles provide multiple binding sites for different cargo and targeting peptides which can be used for development of novel approaches for cancer targeting, diagnostics and therapy. In this review, we will focus on peptides which have been used for preparation of different nanoparticles designed for cancer research.
doi:10.1002/wnan.121
PMCID: PMC3701387  PMID: 21046660
24.  Dual-modality molecular imaging using antibodies labeled with activatable fluorescence and a radionuclide for specific and quantitative targeted cancer detection 
Bioconjugate chemistry  2009;20(11):2177-2184.
Multimodality molecular imaging should have potential for compensating the disadvantages and enhancing the advantages of each modality. Nuclear imaging is superior to optical imaging in whole body imaging and in quantification due to good tissue penetration of gamma rays. However, target specificity can be compromised by high background signal due to the always signal ON feature of nuclear probes. In contrast, optical imaging can be superior in target specific imaging by employing target-specific signal activation systems, although it is not quantitative because of signal attenuation. In this study, to take advantage of the mutual cooperation of each modality, multimodality imaging was performed by a combination of quantitative radiolabeled probe and an activatable optical probe. The monoclonal antibodies, panitumumab (anti-HER1) and trastuzumab (anti-HER2) were labeled with 111In and ICG, and tested in both HER1 and HER2 tumor bearing mice by the cocktail injection of radiolabeled and optical probes, and by the single injection of a dual-labeled probe. The optical and nuclear images were obtained over 6 days after the conjugates injection. The fluorescence activation properties of ICG labeled antibodies were also investigated by in vitro microscopy. In vitro microscopy demonstrated that there was no fluorescence signal with either panitumumab-ICG or trastuzumab-ICG, when the probes were bound to cell surface antigens but were not yet internalized. After the conjugates were internalized into the cells, both conjugates showed bright fluorescence signal only in the target cells. These results show both conjugates work as activatable probes. In vivo multimodality imaging by injection of a cocktail of radio-optical probes, only the target specific tumor was visualized by optical imaging. Meanwhile, the biodistribution profile of the injected antibody was provided by nuclear imaging. Similar results were obtained with radio and optical dual labeled probe, and it is confirmed that pharmacokinetic properties did not affect the results above.
Here, we could characterize the molecular targets by activatable optical probes, and visualize the delivery of targeting molecules quantitatively by radioactive probes. Multimodality molecular imaging combining activatable optical and radioactive probe has great potential for simultaneous visualization, characterization, and measurement of biological processes.
doi:10.1021/bc900362k
PMCID: PMC2782620  PMID: 19919110
25.  Aptamers Generated from Cell-SELEX for Molecular Medicine : A Chemical Biology Approach 
Accounts of chemical research  2010;43(1):48-57.
Conspectus
Molecular medicine is an emerging field focused on understanding the molecular basis of diseases and translating this information into strategies for diagnosis and therapy. This approach could lead to personalized medical treatments. Currently, our ability to understand human diseases at the molecular level is limited by the lack of molecular tools to identify and characterize the distinct molecular features of the disease state, especially for diseases such as cancer. Among the new tools being developed by researchers including chemists, engineers and other scientists is a new class of nucleic acid probes called aptamers, which are ssDNA/RNA molecules selected to target a wide range of molecules and even cells. In this Account, we will focus on the use of aptamers, generated from cell-based selections, as a novel molecular tool for cancer research.
Cancers originate from mutations of human genes. These genetic alterations result in molecular changes to diseased cells, which, in turn, lead to changes in cell morphology and physiology. For decades, clinicians have diagnosed cancers primarily based on the morphology of tumor cells or tissues. However, this method does not always give an accurate diagnosis and does not allow clinicians to effectively assess the complex molecular alterations that are predictive of cancer progression. Although genomics and proteomics do not yet allow a full access to this molecular knowledge, aptamer probes represent one effective and practical avenue toward this goal. One special feature of aptamers is that we can isolate them by selection against cancer cells without prior knowledge of the number and arrangement of proteins on the cellular surface. These probes can identify molecular differences between normal and tumor cells and can discriminate among tumor cells of different classifications, at different disease stages, or from different patients.
This Account summarizes our recent efforts to develop aptamers through cell-SELEX for the study of cancer and apply those aptamers in cancer diagnosis and therapy. We first discuss how we select aptamers against live cancer cells. We then describe uses of these aptamers. Aptamers can serve as agents for molecular profiling of specific cancer types. They can also be used to modify therapeutic reagents to develop targeted cancer therapies. Aptamers are also aiding the discovery of new cancer biomarkers through the recognition of membrane protein targets. Importantly, we demonstrate how molecular assemblies can integrate the properties of aptamers and, for example, nanoparticles or microfluidic devices, to improve cancer cell enrichment, detection and therapy.
doi:10.1021/ar900101s
PMCID: PMC2808443  PMID: 19751057

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