Proceedings Volume 8233

Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications IV

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Proceedings Volume 8233

Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications IV

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Volume Details

Date Published: 16 March 2012
Contents: 12 Sessions, 31 Papers, 0 Presentations
Conference: SPIE BiOS 2012
Volume Number: 8233

Table of Contents

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Table of Contents

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  • Front Matter: Volume 8233
  • Molecular Probes for Targeted Imaging and Therapy
  • NIR Probes for Molecular Imaging
  • Fluorescence Foundations for Probing and Imaging
  • Fluorescence: Lifetime Imaging and Spectroscopy
  • Imaging Molecular Processes with Fluorescent Reporters
  • Multiphoton Imaging Probes
  • Organic Nano Particles for Biomedical Imaging
  • Gold Nanoparticles for Molecular Imaging
  • Inorganic Nanoparticles for Biological Applications
  • Fluorescent Biosensors and Methods
  • Poster Session
Front Matter: Volume 8233
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Front Matter: Volume 8233
This PDF file contains the front matter associated with SPIE Proceedings Volume 8233, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.
Molecular Probes for Targeted Imaging and Therapy
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Electrospray of multifunctional microparticles for image-guided drug delivery
Leilei Zhang, Yan Yan, Joshua Mena, et al.
Anti-VEGF therapies have been widely explored for the management of posterior ocular disease, like neovascular age-related macular degeneration (AMD). Loading anti-VEGF therapies in biodegradable microparticles may enable sustained drug release and improved therapeutic outcome. However, existing microfabrication processes such as double emulsification produce drug-loaded microparticles with low encapsulation rate and poor antibody bioactivity. To overcome these limitations, we fabricate multifunctional microparticles by both single needle and coaxial needle electrospray. The experimental setup for the process includes flat-end syringe needles (both single needle and coaxial needle), high voltage power supplies, and syringe pumps. Microparticles are formed by an electrical field between the needles and the ground electrode. Droplet size and morphology are controlled by multiple process parameters and material properties, such as flow rate and applied voltage. The droplets are collected and freezing dried to obtain multifunctional microparticles. Fluorescent beads encapsulated poly(DL-lactide-co-glycolide) acid (PLGA) microparticles are injected into rabbits eyes through intravitreal injection to test the biodegradable time of microparticles.
Cancer therapy utilizing molecular layer deposition (MLD) and self-organized lightwave network (SOLNET): proposal and theoretical prediction
Tetsuzo Yoshimura, Chie Yoshino, Koyo Sasaki, et al.
Cancer therapy utilizing Molecular Layer Deposition (MLD) and Self-Organized Lightwave Network (SOLNET) is proposed. MLD is a growth method, in which different kinds of molecules are sequentially provided to a substrate to synthesize organic tailored materials with designated molecular arrangements. In cancer therapy, the liquid-phase MLD (LP-MLD) is used with regarding the human body as the MLD chamber and the cancer cells as the substrates. The first proposal is the selective delivery of multi-functional materials with imaging, sensitizing, paramagnetic, lightabsorbing agents etc. to cancer cells by LP-MLD. The second proposal is in-situ synthesis of drugs, especially large and toxic ones, at cancer cells by LP-MLD to deliver the drugs deep inside the cancer without attacking normal cells. The third proposal is the SOLNET-assisted laser surgery. After luminescent molecules are adsorbed in cancer cells by LPMLD, a write beam is introduced from an optical fiber into the area containing cancer cells through photo-induced refractive index increase materials to construct self-aligned optical waveguides of SOLNET, which selectively guides surgery beams to cancer cells. Theoretical predictions and preliminary experimental results are presented.
NIR Probes for Molecular Imaging
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Novel water soluble NIR dyes: does charge matter?
Gabor Patonay, Maged Henary, Garfield Beckford, et al.
Near-Infrared (NIR) dyes are used as reporters, probes or markers in the biological and medical field. NIR dyes can be useful for investigating and characterizing biomolecular interactions or imaging which is possible because biological mammalian tissue has a low absorption window in the NIR region. Biomolecules such as proteins are known to bind to NIR dyes. Upon binding NIR dyes often exhibit spectral changes that can be used for characterizing the binding event. Serum albumins may be responsible for in vivo transport of NIR dyes. Studying this binding event can be useful when correlated to in vivo behavior of the NIR dye. The studies presented here use spectroscopic methods to investigate how NIR dyes that may be used in imaging, biological or bioanalytical applications bind to proteins, such as serum albumins. Our research group systematically synthesized several NIR dyes that have varying hydrophobicity, chromophore size and charge. During these investigations we developed novel NIR cyanine fluorophores having varying aqueous solubility and a variety of net charges. The binding properties of the carbocyanines change when charged or hydrophobic moieties are systematically varied. One of the properties we put a special emphasis on is what we call residual hydrophobicity of the NIR dye molecule which is defined as the unmasked (by the charged moieties) hydrophobicity of the molecule. Residual hydrophobicity may be responsible for binding the otherwise highly water soluble NIR dye to hydrophobic pockets of biomolecules. High residual hydrophobicity of a highly water soluble dye can be disadvantageous during biological, medical or similar applications.
Advancing with pyrrolopyrrole cyanines: a next generation class of near-infrared fluorophores
S. Wiktorowski, G. M. Fischer, E. Daltrozzo, et al.
Fluorescent dyes are the basis for a broad range of modern techniques in life and material sciences. Consequently, there is a pressing need for the development of new classes of NIR fluorophores in recent years. Pyrrolopyrrole Cyanines (PPCys) are a novel class of NIR chromophores that were first presented in 2007 by Fischer and coworkers.[1] Their optical properties are marked by strong and narrowband NIR absorptions, strong NIR fluorescence and hardly any absorption in the visible range. The absorption maxima can be tuned over a broad range while high fluorescence quantum yields are maintained. PPCys are attractive candidates for labelling applications or as selective NIR absorbers. Moreover, PPCys exhibit very high photostability. Due to these outstanding photophysical properties, PPCys are heading into a promising future as NIR dyes.
Fluorescence Foundations for Probing and Imaging
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Fluorescent molecular probes based on excited state prototropism in lipid bilayer membrane
Monalisa Mohapatra, Ashok K. Mishra
Excited state prototropism (ESPT) is observed in molecules having one or more ionizable protons, whose proton transfer efficiency is different in ground and excited states. The interaction of various ESPT molecules like naphthols and intramolecular ESPT (ESIPT) molecules like hydroxyflavones etc. with different microheterogeneous media have been studied in detail and excited state prototropism as a probe concept has been gaining ground. The fluorescence of different prototropic forms of such molecules, on partitioning to an organized medium like lipid bilayer membrane, often show sensitive response to the local environment with respect to the local structure, physical properties and dynamics. Our recent work using 1-naphthol as an ESPT fluorescent molecular probe has shown that the incorporation of monomeric bile salt molecules into lipid bilayer membranes composed from dipalmitoylphosphatidylcholine (DPPC, a lung surfactant) and dimyristoylphosphatidylcholine (DMPC), in solid gel and liquid crystalline phases, induce appreciable wetting of the bilayer up to the hydrocarbon core region, even at very low (≤ 1 mM) concentrations of the bile salts. The incorporation and location of fisetin, an ESIPT molecule having antioxidant properties, in lipid bilayer membrane has been sensitively monitored from its intrinsic fluorescence behaviour.
Iridium complex probes for monitoring of cellular oxygen levels and imaging of hypoxic tissues
Toshitada Yoshihara, Atsushi Kobayashi, Shinpei Oda, et al.
We have recently reported that a red-emitting iridium complex (btp)2 Ir (acac) (BTP) serves as a hypoxia-sensing probe for tumor imaging in living mice. BTP exhibits oxygen-sensitive phosphorescence that can be utilized to monitor oxygen levels in living cells and to visualize hypoxic tissues. To improve the tissue penetrance of BTP, we designed and synthesized near-IR emitting iridium complexes by two different approaches: extension of the π- conjugated system of benzothienyl-pyridinato ligand in BTP and introduction of substituents into suitable positions of ligands. The former approach was successful, and near-IR emitting iridium complexes were obtained without reduction in the emission quantum yield. Cellular uptake of BTP was greatly improved by introducing a hydrophilic group into the acetylacetonato ligand. Using these improved probes, in-vivo lifetime measurements were made to substantiate the hypoxia of tumor tissues in SCC-7 tumor-bearing mice. The second-harmonic (532 nm) of Nd3+:YAG laser was used to excite iridium complexes in tissues, and the phosphorescence lifetime was measured using the time-correlated single photon counting technique. The phosphorescence emitted from the tumor region actually gave longer lifetimes compared to those emitted from the normal tissues, demonstrating the hypoxic nature of tumor tissues.
Amyloid diagnostics: probing protein aggregation and conformation with ultrasensitive fluorescence detection
Rajiv Abhyankar, Banakanidhi Sahoo, Niraj K. Singh, et al.
While dozens of human ailments are now identified as "protein aggregation diseases", aggregation by itself does not seem to be a clear determinant of the toxicity. The structural transformation that accompanies the initial steps of aggregation may be an even more important aspect controlling the biological effects of these protein particles. For this, the key is to develop appropriate fluorescent biomarkers which can probe both aggregation and conformation at low physiological concentrations. Using Alzheimer's amyloid beta (Aβ) as a model system, we have developed probes suitable for the application of Fluorescence Correlation Spectroscopy (FCS, which reports aggregation) and Förster Resonance Energy Transfer (FRET, which reports conformational changes) techniques. To diagnose these changes in the cerebrospinal fluid of Alzheimer's patients, we are now designing better single molecule detection devices. Here we report a confocal device with a 4π collection geometry, which detects more than 0.5 million photons per second from a single rhodamine B molecule in aqueous solution, which to our knowledge is the highest sensitivity achieved so far with such devices. This allows us to perform quick and sensitive antibunching measurements which report the aggregate mass and fluorophore lifetime of Aβ oligomers.
Fluorescence: Lifetime Imaging and Spectroscopy
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Time-domain imaging with quench-based fluorescent contrast agents
Walter J. Akers, Metasebya Solomon, Gail P. Sudlow, et al.
Quench-based probes utilize unique characteristics of fluorescence resonance energy transfer (FRET) to enhance contrast upon de-quenching. This mechanism has been used in a variety of molecular probes for imaging of cancer related enzyme activity such as matrix metalloproteinases, cathepsins and caspases. While non-fluorescent upon administration, fluorescence can be restored by separation of donor and acceptor, resulting in higher intensity in the presence of activator. Along with decreased quantum yield, FRET also results in altered fluorescence lifetime. Time-domain imaging can further enhance contrast and information yield from quench-based probes. We present in vivo time-domain imaging for detecting activation of quench-based probes. Quench-based probes utilize unique characteristics of fluorescence resonance energy transfer (FRET) to enhance contrast upon de-quenching. This mechanism has been used in a variety of molecular probes for imaging of cancer related enzyme activity such as matrix metalloproteinases, cathepsins and caspases. While non-fluorescent upon administration, fluorescence can be restored by separation of donor and acceptor, resulting in higher intensity in the presence of activator. Along with decreased quantum yield, FRET also results in altered fluorescence lifetime. Time-domain imaging can further enhance contrast and information yield from quench-based probes. We present in vivo time-domain imaging for detecting activation of quench-based probes. Time-domain diffuse optical imaging was performed to assess the FRET and quenching in living mice with orthotopic breast cancer. Tumor contrast enhancement was accompanied by increased fluorescence lifetime after administration of quenched probes selective for matrix metalloproteinases while no significant change was observed for non-quenched probes for integrin receptors. These results demonstrate the utility of timedomain imaging for detection of cancer-related enzyme activity in vivo.
Imaging Molecular Processes with Fluorescent Reporters
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Effect of capsid proteins to ICG mass ratio on fluorescent quantum yield of virus-resembling optical nano-materials
Sharad Gupta, Gerardo Ico, Paul Matsumura, et al.
We recently reported construction of a new type of optical nano-construct composed of genome-depleted plant infecting brome mosaic virus (BMV) doped with Indocyanine green (ICG), an FDA-approved chromophore. We refer to these constructs as optical viral ghosts (OVGs) since only the capsid protein (CP) subunits of BMV remain to encapsulate ICG. To utilize OVGs as effective nano-probes in fluorescence imaging applications, their fluorescence quantum yield needs to be maximized. In this study, we investigate the effect of altering the CP to ICG mass ratio on the fluorescent quantum yield of OVGs. Results of this study provide the basis for construction of OVGs with optimal amounts of CP and ICG to yield maximal fluorescence quantum yield.
Imaging B. anthracis heme catabolism in mice using the IFP1.4 gene reporter
Banghe Zhu, Holly Robinson, Nathaniel Wilganowski, et al.
B. anthracis is a gram-positive, spore-forming bacterium which likes all pathogenic bacteria, survive by sequestering heme from its host. To image B. anthracis heme catabolism in vivo, we stably transfect new red excitable fluorescent protein, IFP1.4, that requires the heme catabolism product biliverdin (BV). IFP1.4 reporter has favorable excitation and emission characteristics, which has an absorption peak at 685 nm and an emission peak at 708 nm. Therefore, IFP1.4 reporter can be imaged deeply into the tissue with less contamination from tissue autofluorescence. However, the excitation light "leakage" through optical filters can limit detection and sensitivity of IFP1.4 reporter due to the small Stoke's shift of IFP1.4 fluorescence. To minimize the excitation light leakage, an intensified CCD (ICCD) based infrared fluorescence imaging device was optimized using two band pass filters separated by a focus lens to increase the optical density at the excitation wavelength. In this study, a mouse model (DBA/J2) was first injected with B. anthracis bacteria expressing IFP1.4, 150 μl s.c., on the ventral side of the left thigh. Then mouse was given 250 μl of a 1mM BV solution via I.V. injection. Imaging was conducted as a function of time after infection under light euthanasia, excised tissues were imaged and IFP1.4 fluorescence correlated with standard culture measurements of colony forming units (CFU). The work demonstrates the use of IFP1.4 as a reporter of bacterial utilization of host heme and may provide an important tool for understanding the pathogenesis of bacterial infection and developing new anti-bacterial therapeutics.
Development of anti-HER2 conjugated ICG-loaded polymeric nanoparticles for targeted optical imaging of ovarian cancer
Baharak Bahmani, Valentine Vullev, Bahman Anvari
Targeted delivery of therapeutic and imaging agents using surface modified nanovectors has been explored immensely in recent years. The growing demand for site-specific and efficient delivery of nanovectors entails stable surface conjugation of targeting moieties. We have developed a polymeric nanocapsule doped with Indocyanine green (ICG) with potential for targeted and deep tissue optical imaging and phototherapy. Our ICG-loaded nanocapsules (ICG-NCs) have potential for covalent coupling of various targeting moieties and materials due to presence of amine groups on the surface. Here, we covalently bioconjugate polyethylene glycol(PEG)-coated ICG-NCs with monoclonal antibody against HER2 through reductive amination-mediated procedures. The irreversible and stable bonds are formed between anti- EGFR and aldehyde termini of PEG chains on the surface of ICG-NCs. We confirm the uptake of conjugated ICG-NCs by ovarian cancer cells over-expressing HER2 using fluorescent confocal microscopy. The proposed process for covalent attachment of anti-HER2 to PEGylated ICG-NCs can be used as a methodology for bioconjugation of various antibodies to such nano-constrcuts, and provides the capability to use these optically active nano-probes for targeted optical imaging of ovarian and other cancer types.
Multiphoton Imaging Probes
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In vivo track the development of melanoma with the intrinsic third harmonic generation and two-photon fluorescence contrasts of melanin
Pei-Chun Wu, Yu-Shing Chen, Tsung-Yuan Hsieh, et al.
The understanding of the interaction between tumors and surrounding microenvironment in vivo is an important first step and basis for pathway-targeting cancer therapy. To in vivo observe the dynamic development of tumor cells and validate the efficacy of therapy in microscopic scales, people commonly performed multi-photon fluorescence microscopy through an invasive window chamber setup. However, under such system, the cancer cells can't be identified and long-term tracked without a fluorescence labeling. Exploiting the intrinsic third harmonic generation (THG) and two-photon fluorescence (2PF) contrasts of melanin, we demonstrated in vivo identification of melanoma and tracked its development without labeling. It was achieved with a least invasive femtosecond Cr:forsterite laser and a laser scanning nonlinear microscopy system with 3D sub-micron spatial resolution. Combined with molecular probes or reporters, we anticipate thus developed platform a powerful tool to reveal molecular insights of tumor microenvironments, enhance our understanding of tumor biology, and trigger new therapeutic approaches.
Multiphoton fluorescence spectra and lifetimes of biliverdins and their protein-associated complex
To investigate whether endogenous biliverdins can serve as a fluorescence metabolic marker in cancer diagnosis, we measured their multiphoton fluorescence spectra and lifetimes with femtosecond Cr:forsterite laser. Excited at 1230nm, the two-photon fluorescence of biliverdins peaks around 670nm. The corresponding lifetime (<100ps) was much shorter than those of porphyrins (~10ns), which is another commonly present metabolites in living cells. Further mixing biliverdins with proteins like fetal bovine serum (FBS), biliverdins reductase A (BVRA), or heme oxygenase-1 (HO-1), the yields of red autofluorescences didn't change a lot, but the corresponding lifetimes with HO-1 and BSA were lengthened to 200~300ps. This indicates that biliverdin can have an association with these proteins and change its lifetime. These spectral and temporal characteristics of fluorescence make biliverdin a potential marker fluorophore for hyperspectral diagnosis on the heme catabolism in human cells or tissues.
Detection of amino acid neurotransmitters by surface enhanced Raman scattering and hollow core photonic crystal fiber
Vidhu S. Tiwari, Altaf Khetani, Ali Momenpour T. Monfared, et al.
The present work explores the feasibility of using surface enhanced Raman scattering (SERS) for detecting the neurotransmitters such as glutamate (GLU) and gamma-amino butyric acid (GABA). These amino acid neurotransmitters that respectively mediate fast excitatory and inhibitory neurotransmission in the brain, are important for neuroendocrine control, and upsets in their synthesis are also linked to epilepsy. Our SERS-based detection scheme enabled the detection of low amounts of GLU (10-7 M) and GABA (10-4 M). It may complement existing techniques for characterizing such kinds of neurotransmitters that include high-performance liquid chromatography (HPLC) or mass spectrography (MS). This is mainly because SERS has other advantages such as ease of sample preparation, molecular specificity and sensitivity, thus making it potentially applicable to characterization of experimental brain extracts or clinical diagnostic samples of cerebrospinal fluid and saliva. Using hollow core photonic crystal fiber (HC-PCF) further enhanced the Raman signal relative to that in a standard cuvette providing sensitive detection of GLU and GABA in micro-litre volume of aqueous solutions.
Organic Nano Particles for Biomedical Imaging
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Design of peptide-conjugated glycol chitosan nanoparticles for near infrared fluorescent (NIRF) in vivo imaging of bladder tumors
Jaehong Key, Deepika Dhawan, Deborah W. Knapp, et al.
Enhanced permeability and retention (EPR) effects for tumor treatment have been utilized as a representative strategy to accumulate untargeted nanoparticles in the blood vessels around tumors. However, the EPR effect itself was not sufficient for the nanoparticles to penetrate into cancer cells. For the improvement of diagnosis and treatment of cancer using nanoparticles, many more nanoparticles need to specifically enter cancer cells. Otherwise, can leave the tumor area and not contribute to treatment. In order to enhance the internalization process, specific ligands on nanoparticles can help their specific internalization in cancer cells by receptor-mediated endocytosis. We previously developed glycol chitosan based nanoparticles that suggested a promising possibility for in vivo tumor imaging using the EPR effect. The glycol chitosan nanoparticles showed a long circulation time beyond 1 day and they were accumulated predominantly in tumor. In this study, we evaluated two peptides for specific targeting and better internalization into urinary bladder cancer cells. We conjugated the peptides on to the glycol chitosan nanoparticles; the peptide-conjugated nanoparticles were also labeling with near infrared fluorescent (NIRF) dye, Cy5.5, to visualize them by optical imaging in vivo. Importantly real-time NIRF imaging can also be used for fluorescence (NIRF)-guided surgery of tumors beyond normal optical penetration depths. The peptide conjugated glycol chitosan nanoparticles were characterized with respect to size, stability and zeta-potential and compared with previous nanoparticles without ligands in terms of their internalization into bladder cancer cells. This study demonstrated the possibility of our nanoparticles for tumor imaging and emphasized the importance of specific targeting peptides.
Plant virus-resembling optical nano-materials conjugated with anti-EGFR for targeted cancer imaging
Sharad Gupta, Hailey Wilder, A. L. N. Rao, et al.
We recently reported the construction of a new type of optically active nano-particles composed of genome-depleted plant infecting brome mosaic virus (BMV) doped with indocyanine green (ICG), an FDA-approved chromophore . We refer to these constructs as optical viral ghosts (OVGs) since only the capsid protein (CP) subunits of BMV remain to encapsulate ICG. Herein, we covalently conjugated the surface of OVGs with anti-epidermal growth factor receptors (anti-EGFR) to target cancerous human bronchial epithelial cells (C-HBECs) in-vitro. Our preliminary results demonstrate the utility of conjugated OVGs for targeted imaging of cancer cells.
Targeted polymeric magnetic nanoparticles for brain imaging
Bharat Kirthivasan, Dhirender Singh, Sangram Raut, et al.
The purpose of this study was to develop targeted polymeric magnetic nanoparticle system for brain imaging. Near infrared dye indocyanine green (ICG) or p-gycoprotein substrate rhodamine 123 (Rh123) were encapsulated along with oleic acid coated magnetic nanoparticles (OAMNP) in a matrix of poly(lactide-co-glycolide) (PLGA) and methoxy poly(ethyleneglycol)-poly(lactide) (Met-PEG-PLA). The nanoparticles were evaluated for morphology, particle size, dye content and magnetite content. The in vivo biodistribution study was carried out using three groups of six male Sprague Dawley rats each. Group I received a saline solution containing the dye, group II received dye-loaded polymeric magnetic nanoparticles without the aid of a magnetic field, and group III received dye-loaded polymeric magnetic nanoparticles with a magnet (8000 G) placed on the head of the rat. After a preset exposure period, the animals were sacrificed and dye concentration was measured in the brain, liver, kidney, lungs and spleen homogenates. Brain sections were fixed, cryotomed and visualized using fluorescence microscopy. The particles were observed to be spherical and had a mean size of 220 nm. The encapsulation efficiency for OAMNP was 57%, while that for ICG was 56% and for Rh123 was 45%. In the biodistribution study, while the majority of the dose for all animals was found in the liver, kidneys and spleen, group III showed a significantly higher brain concentration than the other two groups (p < 0.001). This result was corroborated by the fluorescence microscopy studies, which showed enhanced dye penetration into the brain tissue for group III. Further studies need to be done to elucidate the exact mechanism responsible for the increased brain uptake of dye to help us understand if the magnetic nanoparticles actually penetrate the blood brain barrier or merely deliver a massive load of dye just outside it, thereby triggering passive diffusion into the brain parenchyma. These results reinforce the potential use of polymeric magnetically-targeted nanoparticles in active brain targeting and imaging.
Multi-modal in cellulo evaluation of NPR-C targeted C-ANF-peptide and C-ANF-comb nanoparticles
Monica Shokeen, Eric Pressly, Luke Connal, et al.
Natriuretic peptides (NPs) are clinical markers of heart disease that have anti-proliferative and anti-migratory effects on vascular smooth-muscle cells (VSMCs). In atherosclerosis, NPs participate in vascular remodeling, where the expression of NP clearance receptors (NPR-Cs) is upregulated both in the endothelium and in VSMCs[1-3]. In this study, we investigated the enhanced targeting potential of novel multifunctional nanoprobes conjugated with multiple copies of a C-type atrial natriuretic factor (C-ANF) peptide fragment to target NPR-C transfected cells. The cell binding results of the NPR-C targeted nanoprobes were compared with that of the C-ANF peptide fragment alone. The nanoprobe and peptide structures contain the chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) for labeling with the PET tracer, 64Cu, for radioactive assays and luminescent Eu (III) for confocal cell imaging. Cell assays performed with the radioactive nanoprobe and peptide demonstrated higher cell binding of the targeted nanoprobe comapred with the peptide alone (8.63±1.67 vs. 1.13±0.06). The targeting specificity of both moieties was tested by using the control cell lines NPR-A and NPR-B, and receptor mediated uptake was demonstrated by reduced uptake in the presence of excess unlabeled respective probes.
Gold Nanoparticles for Molecular Imaging
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Calibrating the imaging and therapy performance of magneto-fluorescent gold nanoshells for breast cancer
Adam Dowell, Wenxue Chen, Nrusingh Biswal, et al.
Gold nanoshells with NIR plasmon resonance can be modified to simultaneously enhance conjugated NIR fluorescence dyes and T2 contrast of embedded iron-oxide nanoparticles, and molecularly targeted to breast and other cancers. We calibrated the theranostic performance of magneto-fluorescent nanoshells, and contrasted the performance of molecularly targeted and untargeted nanoshells for breast cancer therapy, employing MCF-7L and their HER2 overexpressing derivative MCF-7/HER2-18 breast cancer cells as in vitro model systems. Silica core gold nanoshells with plasmon resonance on ~810 nm were doped with NIR dye ICG and ~10 nm iron-oxide nanoparticles in a ~20 nm epilayer of silica. A subset of nanoshells was conjugated to antibodies targeting HER2. Cell viability with varying laser power levels in presence and absence of bare and HER2-targeted nanoshells was assessed by calcein and propidium iodide staining. For MCF-7L cells, increasing power resulted in increased cell death (F=5.63, p=0.0018), and bare nanoshells caused more cell death than HER2-targeted nanoshells or laser treatment alone (F=30.13, p<0.001). For MCF-7/HER2-18 cells, death was greater with HER2-targeted nanoshells and was independent of laser power. This study demonstrates the capability of magneto-fluorescent nanocomplexes for imaging and therapy of breast cancer cells, and the advantages of targeting receptors unique to cancer cells.
Inorganic Nanoparticles for Biological Applications
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Self-illuminating nanoprobe for in vivo imaging of cancers over-expressing the folate receptor
Steven C. Miller, Lucia Beviglia, Pete Yeung, et al.
New in vivo imaging reagents with increased sensitivity and penetration depth are needed to advance our understanding of metastases and accelerate the development of therapeutics. The folate receptor (FR) is a promising imaging target that is up-regulated in many human carcinomas, including cancers of the ovary, breast, pancreas, endometrium, lungs, kidneys, colon, brain, and myeloid cells. Zymera has developed a self-illuminating Bioluminescence Resonance Energy Transfer Quantum Dot (BRET-Qdot) nanoprobe conjugated with folate (BQ-Folate) for in vivo imaging of cancers overexpressing FR. BQ-Folate is a novel nanoprobe formed by co-conjugating Renilla reniformis luciferase enzyme and folate to near-infrared (NIR) emitting quantum dots. The luciferase substrate, coelenterazine, activates the BQ-Folate nanoprobe generating luminescence emission in the near-infrared (NIR) region (655 nm) for increased sensitivity and penetration depth. Because BQ-Folate requires no external light source for light emission, it has significant advantages for challenging in vivo preclinical optical imaging applications, such as the detection of early stage metastases. Zymera and OncoMed Pharmaceuticals have demonstrated that in vivo imaging with the BQ-Folate nanoprobe detected the primary tumor and early stage metastases in an orthotopic NOD/SCID mouse model of human pancreatic cancer.
Dual modality photothermal OCT and magnetic resonance imaging with carbon nanotubes
Jason M. Tucker-Schwartz, Tu Hong, Daniel C. Colvin, et al.
Preclinical molecular imaging of cancer has the potential to increase the understanding of fundamental cancer biology, elucidate mechanisms of cancer treatment resistance, and increase effectiveness of drug candidates. Optical and magnetic resonance imaging contain complementary strengths, suitable for gaining a wealth of knowledge when combined. Here, we demonstrate the inherent contrast sensitivity of single walled carbon nanotubes to absorption based photothermal optical coherence tomography (PT-OCT), and magnetic resonance imaging spin dephasing contrast (T2). A spectral-domain OCT system was interfaced with an amplitude-modulated (100 Hz) titanium sapphire pump beam for PT-OCT imaging. MRI was performed with a commercial 4.7 T animal scanner. With both imaging tools, contrast agent signal linearity (r2 > 0.95) and nM sensitivity over background (p < 0.05) was experimentally determined with serially dilute solutions of carbon nanotubes coated in amine-terminated polyethylene glycol. The surface functionalization chemistry for carbon nanotubes is well understood, and molecular targeting has been demonstrated in vitro and in vivo, making carbon nanotubes an attractive agent for molecular imaging in preclinical models. We have demonstrated the initial characterization steps for using carbon nanotubes for multi-modality imaging with PT-OCT and MRI.
Super strong nanoindentors for biomedical applications based on bamboo-like nanotubes
O. E. Glukhova, A. S. Kolesnikova, E. L. Kossovich, et al.
The results of quantum-chemical analysis of elastic and strength properties of the bamboo-like tube are presented in this paper. For the first time the configuration of the thinnest stable bamboo-like tube was established. The bamboo-like nanotube breaking point is established to be under compression of 11GPa. Configuration of the nanoindentor based on symmetric and streamlined tip of the tube (15,15), presented in this work, provides perfect interaction between the nanoindentor tip and the tissue because tip has no sharp protruding pieces.
Highly efficient phosphors in cancer sensing and PDT
Brian G. Yust, Lawrence C. Mimun, Dhiraj K. Sardar, et al.
Highly efficient upconverting phosphors (NaYF4) doped with erbium ions are bio-conjugated and used for cancer imaging and photodynamic therapy. Once they are conjugated, the particles are injected into mice to demonstrate that cancer imaging with a near-infrared excitation source is possible. Finally, the particles are also conjugated with a photosensitive molecule with strong absorption near the upconversion emission peak (~ 550nm). The upconversion energy causes the photosensitive molecule to create highly reactive oxidative species, which puncture and kill the cell to which it is attached. These particles are then used in a mouse model, and the size of the tumors is modeled as a function of the dosage and duration of the photodynamic therapy.
Fluorescent Biosensors and Methods
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The curvature influence of the graphene nanoribbon on its sensory properties
O. E. Glukhova, I. V. Kirillova, M. M. Slepchenkov
The results of the theoretical investigation of the curvature influence of the strained graphene nanoribbon on its sensory properties are presented in the given work. The attachment mechanisms of hydrogen atoms to the plane and the wavelike graphene nanoribbon are studied by the tight-binding method. For the first time it was established, that the sensory properties of nanoribbon improve with increase of the surface curvature. It was revealed, that the potential well depth of interaction of the curved graphene with hydrogen atom is greater than the planar graphene. It was established, that the difference of the potential minima of the C-H interaction energy increases exponentially with the curvature increase.
New cross-linking quinoline- and quinolone-based luminescent lanthanide probes for sensitive labeling
Shyamala Pillai, Laura Wirpsza, Maxim Kozlov, et al.
New luminescent lanthanide chelates containing thiol-, amine-, and click-reactive groups in antenna-fluorophore moieties were synthesized. The chelates include diethylenetriaminepentaacetic acid (DTPA) coupled to two types of chromophores: 7-amino-4-trifluoromethyl-2(1H) quinolinone, and 7-amino-4-trifluoromethyl-2-alkoxyquinoline. The synthesized compounds were characterized using NMR, light absorption, steady-state and time-resolved fluorescent spectroscopy. Some of the compounds displayed high brightness with Tb3+, Eu3+, and Dy3+. Obtained reactive lanthanide chelates can be easily attached to biological molecules. The probes demonstrated high performance in molecular beaconbased DNA hybridization assays (sub-pico molar detection limit), in bacterial proteome labeling, and in live cell imaging.
Poster Session
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Water participation in molecular recognition and protein-ligand association: Probing the drug binding site "Sudlow I" in human serum albumin
Najla Al-Lawatia, Thomas Steinbrecher, Osama K. Abou-Zied
Human serum albumin (HSA) plays an important role in the transport and disposition of endogenous and exogenous ligands present in blood. Its capacity to reversibly bind a large variety of drugs results in its prevailing role in drug pharmacokinetics and pharmacodynamics. In this work, we used 7-hydroxyquinoline (7HQ) as a probe to study the binding nature of one of the major drug binding sites of HSA (Sudlow I) and to reveal the local environment around the probe in the binding site. The interaction between 7HQ and HSA at a physiological pH of 7.2 was investigated using steady-state and lifetime spectroscopic measurements, molecular docking and molecular dynamics (MD) simulations methods. The fluorescence results indicate a selective interaction between 7HQ and the Trp214 residue. The reduction in both the intensity and lifetime of the Trp214 fluorescence upon probe binding indicates the dominant role of static quenching. Molecular docking and MD simulations show that 7HQ binds in Sudlow site I close to Trp214, confirming the experimental results, and pinpoint the dominant role of hydrophobic interaction in the binding site. Electrostatic interactions were also found to be important in which two water molecules form strong hydrogen bonds with the polar groups of 7HQ. Detection of water in the binding site agrees with the absorption and fluorescence results that show the formation of a zwitterion tautomer of 7HQ. The unique spectral signatures of 7HQ in water make this molecule a potential probe for detecting the presence of water in nanocavities of proteins. Interaction of 7HQ with water in the binding site shows that water molecules can be crucial for molecular recognition and association in protein binding sites.
Strain-hardening effect of graphene on a chitosan chain for the tissue engineering
O. E. Glukhova, I. V. Kirillova, A. S. Kolesnikova, et al.
We report the results of the chitosan dimer study, the mechanism of its interaction with the carbon nanostructures and also the mechanical properties of the chitosan/graphene, chitosan/nanotube complexes using the density function and the molecular dynamic methods. It was established that the physical adsorption of the chitosan with graphene is carried out by the Van der Waals interaction between the hexagonal links of the chitosan with the hexagonal cell of the atomic grid of graphene and nanotube.
The observation research of the differences in cell death and reactive oxygen species in the process of infecting Arabidopsis with avirulent strains
HuaBin Liu, WenLi Chen
Objective: To observe the differences of cell death and accumulation of reactive oxygen species (ROS) in the process of infecting Arabidopsis with avirulent Pseudomonas syringae pv. tomato DC3000 (avrB, avrRps4), it will be of great importance to research the role of plant disease resistance and defense response. Methods: Using WT, AtrbohD and AtrbohF mutant as experimental materials, we discuss the impact of cell death and ROS on the leaves of Arabidopsis infected with avirulent Pst DC3000 (avrB, avrRps4), observed by spectral analysis and visualized by DAB and trypan blue stain. Results: When infected with avirulent Pst DC3000, both WT and AtrbohF mutant line behaved resistance that exhibited burst of ROS and HR occur, limit senescence and pathogen induced chlorotic cell death. Paradoxically, AtrbohD mutant behaved susceptible characters that exhibited a small quantity of ROS accumulated and enhanced cell death. Conclusion: After infection of Arabidopsis with avirulent Pst DC3000, WT exhibited more ROS accumulation than AtrbohF, and AtrbohD eliminated the majority of total ROS production. Although both WT and AtrbohF mutant exhibited HR occur, enhanced cell death in AtrbohD mutant.
The observation of mitochondrial movement and ATG5 position in Arabidopsis during the process of infection with virulent and avirulent P. syringae strains
Liu Yang, Chao Ma, Wen li Chen
Infection of plants with pathogens leads to programmed cell death (PCD) associated with the pathogen-triggered hypersensitive response (HR) during plant innate immunity. In this study, the effects of infection by virulent Pseudomonas syringae pv. tomato (Pst) DC3000 and strains harboring avirulence factors AvrRps4 on the induction of HR-PCD were compared. We used Arabidopsis thaliana plants as materials, which expressed green fluorescent protein labeled mitochondria (mito-GFP) and green fluorescent protein tagged ATG5 (ATG5-GFP), these GFP are instantaneous expression. We found both Pst DC3000 and Pst-avrRps4 could induce mitochondria to assemble, the effect of Pst DC3000 was more obvious. ATG5 was located in chloroplasts after infection with Pst DC3000 or Pst-avrRps4. Under the condition of Pst-avrRps4, the expression of ATG5 was stronger than Pst DC3000 treatment.
Fluorescent nanodiamonds as highly stable biomarker for endotoxin verification
Thorsten Bergmann, Jan Michael Burg, Maria Lilholt, et al.
Fluorescent nanodiamonds (ND) provide advantageous properties as a fluorescent biomarker for in vitro and in vivo studies. The maximum fluorescence occurs around 700 nm, they do not show photobleaching or blinking and seem to be nontoxic. After a pretreatment with strong acid fluorescent ND can be functionalized and coupled to endotoxin. Endotoxin is a decay product of bacteria and causes strong immune reactions. Therefore endotoxin has to be removed for most applications. An effective removal procedure is membrane filtration. The endotoxin, coupled to fluorescent ND can be visualized by using confocal microscopy which allows the investigation of the separation mechanisms of the filtration process within the membranes.