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Multimodal Multiplexed Immunoimaging with Nanostars to Detect Multiple Immunomarkers and Monitor Response to Immunotherapies.
Ou YC, Wen X, Johnson CA, Shae D, Ayala OD, Webb JA, Lin EC, DeLapp RC, Boyd KL, Richmond A, Mahadevan-Jansen A, Rafat M, Wilson JT, Balko JM, Tantawy MN, Vilgelm AE, Bardhan R
(2020) ACS Nano 14: 651-663
MeSH Terms: Animals, B7-H1 Antigen, Biomarkers, Tumor, Cell Line, Tumor, Disease Models, Animal, Gold, Immunotherapy, Melanoma, Metal Nanoparticles, Mice, Optical Imaging, Particle Size, Surface Properties, Tumor Necrosis Factor Receptor Superfamily, Member 9
Show Abstract · Added March 17, 2020
The overexpression of immunomarker programmed cell death protein 1 (PD-1) and engagement of PD-1 to its ligand, PD-L1, are involved in the functional impairment of cluster of differentiation 8 (CD8) T cells, contributing to cancer progression. However, heterogeneities in PD-L1 expression and variabilities in biopsy-based assays render current approaches inaccurate in predicting PD-L1 status. Therefore, PD-L1 screening alone is not predictive of patient response to treatment, which motivates us to simultaneously detect multiple immunomarkers engaged in immune modulation. Here, we have developed multimodal probes, immunoactive gold nanostars (IGNs), that accurately detect PD-L1 tumor cells and CD8 T cells simultaneously , surpassing the limitations of current immunoimaging techniques. IGNs integrate the whole-body imaging of positron emission tomography with high sensitivity and multiplexing of Raman spectroscopy, enabling the dynamic tracking of both immunomarkers. IGNs also monitor response to immunotherapies in mice treated with combinatorial PD-L1 and CD137 agonists and distinguish responders from those nonresponsive to treatment. Our results showed a multifunctional nanoscale probe with capabilities that cannot be achieved with either modality alone, allowing multiplexed immunologic tumor profiling critical for predicting early response to immunotherapies.
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14 MeSH Terms
Merging Orthovoltage X-Ray Minibeams spare the proximal tissues while producing a solid beam at the target.
Dilmanian FA, Krishnan S, McLaughlin WE, Lukaniec B, Baker JT, Ailawadi S, Hirsch KN, Cattell RF, Roy R, Helfer J, Kruger K, Spuhler K, He Y, Tailor R, Vassantachart A, Heaney DC, Zanzonico P, Gobbert MK, Graf JS, Nassimi JR, Fatemi NN, Schweitzer ME, Bangiyev L, Eley JG
(2019) Sci Rep 9: 1198
MeSH Terms: Brain Neoplasms, Computer Simulation, Gold, Humans, Metal Nanoparticles, Models, Biological, Monte Carlo Method, Radiography, Radiometry, Radiosurgery, Radiotherapy, Radiotherapy Dosage, X-Ray Therapy, X-Rays
Show Abstract · Added March 30, 2020
Conventional radiation therapy of brain tumors often produces cognitive deficits, particularly in children. We investigated the potential efficacy of merging Orthovoltage X-ray Minibeams (OXM). It segments the beam into an array of parallel, thin (~0.3 mm), planar beams, called minibeams, which are known from synchrotron x-ray experiments to spare tissues. Furthermore, the slight divergence of the OXM array make the individual minibeams gradually broaden, thus merging with their neighbors at a given tissue depth to produce a solid beam. In this way the proximal tissues, including the cerebral cortex, can be spared. Here we present experimental results with radiochromic films to characterize the method's dosimetry. Furthermore, we present our Monte Carlo simulation results for physical absorbed dose, and a first-order biologic model to predict tissue tolerance. In particular, a 220-kVp orthovoltage beam provides a 5-fold sharper lateral penumbra than a 6-MV x-ray beam. The method can be implemented in arc-scan, which may include volumetric-modulated arc therapy (VMAT). Finally, OXM's low beam energy makes it ideal for tumor-dose enhancement with contrast agents such as iodine or gold nanoparticles, and its low cost, portability, and small room-shielding requirements make it ideal for use in the low-and-middle-income countries.
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14 MeSH Terms
Targeted Imaging of VCAM-1 mRNA in a Mouse Model of Laser-Induced Choroidal Neovascularization Using Antisense Hairpin-DNA-Functionalized Gold-Nanoparticles.
Uddin MI, Kilburn TC, Yang R, McCollum GW, Wright DW, Penn JS
(2018) Mol Pharm 15: 5514-5520
MeSH Terms: Animals, Biomarkers, Choroid, Choroidal Neovascularization, Disease Models, Animal, Fluorescent Dyes, Gold, Humans, Intravital Microscopy, Lasers, Male, Metal Nanoparticles, Mice, Mice, Inbred C57BL, Molecular Imaging, Molecular Probes, Oligodeoxyribonucleotides, Antisense, Optical Imaging, RNA, Messenger, Vascular Cell Adhesion Molecule-1, Wet Macular Degeneration
Show Abstract · Added April 10, 2019
Mouse laser-induced choroidal neovascularization (mouse LCNV) recapitulates the "wet" form of human age-related macular degeneration (AMD). Vascular cell adhesion molecule-1 (VCAM-1) is a known inflammatory biomarker, and it increases in the choroidal neovascular tissues characteristic of this experimental model. We have designed and constructed gold nanoparticles (AuNPs) functionalized with hairpin-DNA that incorporates an antisense sequence complementary to VCAM-1 mRNA (AS-VCAM-1 hAuNPs) and tested them as optical imaging probes. The 3' end of the hairpin is coupled to a near-infrared fluorophore that is quenched by the AuNP surface via Förster resonance energy transfer (FRET). Hybridization of the antisense sequence to VCAM-1 mRNA displaces the fluorophore away from the AuNP surface, inducing fluorescent activity. In vitro testing showed that hAuNPs hybridize to an exogenous complementary oligonucleotide within a pH range of 4.5-7.4, and that they are stable at reduced pH. LCNV mice received tail-vein injections of AS-VCAM-1 hAuNPs. Hyperspectral imaging revealed the delivery of AS-VCAM-1 hAuNPs to excised choroidal tissues. Fluorescent images of CNV lesions were obtained, presumably in response to the hybridization of AS-hAuNPs to LCNV-induced VCAM-1 mRNA. This is the first demonstration of systemic delivery of hAuNPs to ocular tissues to facilitate mRNA imaging of any target.
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21 MeSH Terms
Colistin-Functionalized Nanoparticles for the Rapid Capture of Acinetobacter baumannii.
Miller SE, Bell CS, Mejias R, McClain MS, Cover TL, Giorgio TD
(2016) J Biomed Nanotechnol 12: 1806-19
MeSH Terms: Acinetobacter baumannii, Bacteriological Techniques, Cell Separation, Colistin, Gold, Metal Nanoparticles
Show Abstract · Added March 21, 2018
Gold nanoparticles (AuNPs) were functionalized for rapid binding of Acinetobacter baumannii (A. baumannii), a Gram-negative bacterium. AuNPs were functionalized with colistin (Col), a polycationic antibiotic, using a two-step self-assembly process, in which heterobifunctional polyethylene glycol (PEG) was used as a linker. Colistin was successfully conjugated to the AuNPs (Col-PEG-AuNP), as validated by dynamic light scattering (DLS) and proton nuclear magnetic resonance (H1 NMR). High angle annular dark field scanning transmission electron microscopy (HAADF-STEM) images, acquired simultaneously with X-ray energy dispersive spectroscopy (EDS) data, confirmed binding of Col-PEG-AuNPs to the cell envelope of A. baumannii. Results generated from a binding assay indicated that Col-PEG-AuNP complexation with A. baumannii occurred rapidly and reached half-maximum saturation in approximately 7 minutes, on average, for all A. baumannii strains evaluated. Quantitative measurement of the kinetics of Col-PEG-AuNP binding to A. baumannii is essential to inform the design of colistin-functionalized magnetic nanoparticles for magnetic separation of nanoparticle-bound A. baumannii.
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6 MeSH Terms
Real-time imaging of VCAM-1 mRNA in TNF-α activated retinal microvascular endothelial cells using antisense hairpin-DNA functionalized gold nanoparticles.
Uddin MI, Jayagopal A, Wong A, McCollum GW, Wright DW, Penn JS
(2018) Nanomedicine 14: 63-71
MeSH Terms: Animals, Cell Survival, Cells, Cultured, DNA, Antisense, Endothelium, Vascular, Fluorescence, Gold, Metal Nanoparticles, Mice, Molecular Imaging, RNA, Messenger, Retinal Vessels, Tumor Necrosis Factor-alpha, Vascular Cell Adhesion Molecule-1
Show Abstract · Added December 21, 2017
Vascular cell adhesion molecule 1 (VCAM-1) is an important inflammatory biomarker correlating with retinal disease progression. Thus, detection of VCAM-1 mRNA expression levels at an early disease stage could be an important predictive biomarker to assess the risk of disease progression and monitoring treatment response. We have developed VCAM-1 targeted antisense hairpin DNA-functionalized gold nanoparticles (AS-VCAM-1 hAuNP) for the real time detection of VCAM-1 mRNA expression levels in retinal endothelial cells. The AS-VCAM-1 hAuNP fluorescence enhancement clearly visualized the TNF-α induced cellular VCAM-1 mRNA levels with high signal to noise ratios compared to normal serum treated cells. The scrambled hAuNP probes were minimally detectable under same image acquisition conditions. Intracellular hAuNPs were detected using transmission electron microscopy (TEM) analysis of the intact cells. In addition, the AS-VCAM-1 hAuNP probes exhibited no acute toxicity to the retinal microvascular endothelial cells as measured by live-dead assay.
Copyright © 2017 Elsevier Inc. All rights reserved.
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14 MeSH Terms
In vivo testing for gold nanoparticle toxicity.
Simpson CA, Huffman BJ, Cliffel DE
(2013) Methods Mol Biol 1026: 175-86
MeSH Terms: Animals, Blood Chemical Analysis, Blood Specimen Collection, Erythrocyte Count, Female, Gold, Leukocyte Count, Metal Nanoparticles, Mice, Mice, Inbred BALB C, Polyethylene Glycols, Species Specificity, Tissue and Organ Harvesting, Toxicity Tests, Urinalysis, Urine Specimen Collection
Show Abstract · Added January 20, 2015
A technique for measuring the toxicity of nanomaterials using a murine model is described. Blood samples are collected via submandibular bleeding while urine samples are collected on cellophane sheets. Both biosamples are then analyzed by inductively coupled plasma optical emission spectroscopy (ICP-OES) for nanotoxicity. Blood samples are further tested for immunological response using a standard Coulter counter. The major organs of interest for filtration are also digested and analyzed via ICP-OES, producing useful information regarding target specificity of the nanomaterial of interest. Collection of the biosamples and analysis afterward is detailed, and the operation of the technique is described and illustrated by analysis of the nanotoxicity of an injection of a modified tiopronin monolayer-protected cluster.
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16 MeSH Terms
Detecting respiratory syncytial virus using nanoparticle-amplified immuno-PCR.
Perez JW, Adams NM, Zimmerman GR, Haselton FR, Wright DW
(2013) Methods Mol Biol 1026: 93-110
MeSH Terms: Antibodies, Viral, Cell Line, DNA, Viral, Gold, Immunoassay, Limit of Detection, Magnets, Metal Nanoparticles, Microspheres, Quartz Crystal Microbalance Techniques, RNA, Viral, Real-Time Polymerase Chain Reaction, Reproducibility of Results, Respiratory Syncytial Viruses
Show Abstract · Added May 27, 2014
Early-stage detection is essential for effective treatment of pediatric virus infections. In traditional -immuno-PCR, a single antibody recognition event is associated with one to three DNA tags, which are subsequently amplified by PCR. In this protocol, we describe a nanoparticle-amplified immuno-PCR assay that combines antibody recognition of traditional ELISA with a 50-fold nanoparticle valence amplification step followed by amplification by traditional PCR. The assay detects a respiratory syncytial virus (RSV) surface fusion protein using a Synagis antibody bound to a 15 nm gold nanoparticle co-functionalized with thiolated DNA complementary to a hybridized 76-base Tag DNA. The Tag DNA to Synagis ratio is 50 to 1. The presence of virus particles triggers the formation of a "sandwich" complex comprised of the gold nanoparticle construct, virus, and a 1 μm antibody-functionalized magnetic particle used for extraction. Virus-containing complexes are isolated using a magnet, DNA tags released by heating to 95 °C, and detected via real-time PCR. The limit of detection of the nanoparticle-amplified immuno-PCR assay was compared to traditional ELISA and traditional RT-PCR using RSV-infected HEp-2 cell extracts. Nanoparticle-amplified immuno-PCR showed a ∼4,000-fold improvement in the limit of detection compared to ELISA and a fourfold improvement in the limit of detection compared to traditional RT-PCR. Nanoparticle-amplified immuno-PCR offers a viable platform for the development of an early-stage diagnostics requiring an exceptionally low limit of detection.
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14 MeSH Terms
Three-dimensional molecular imaging with photothermal optical coherence tomography.
Skala MC, Crow MJ, Wax A, Izatt JA
(2013) Methods Mol Biol 1026: 85-92
MeSH Terms: Cell Line, Tumor, Darkness, Gold, Hot Temperature, Humans, Imaging, Three-Dimensional, Immunoconjugates, Metal Nanoparticles, Molecular Imaging, Tissue Culture Techniques, Tomography, Optical Coherence
Show Abstract · Added March 11, 2014
Optical coherence tomography (OCT) is a three-dimensional optical imaging technique that has been successfully implemented in ophthalmology for imaging the human retina, and in studying animal models of disease. OCT can nondestructively visualize structural features in tissue at cellular-level resolution, and can exploit contrast agents to achieve molecular contrast. Photothermal OCT relies on the heat-producing capabilities of antibody-conjugated gold nanoparticles to achieve molecular contrast. A pump laser at the nanoparticle resonance wavelength is used to heat the nanoparticles in the sample, and the resulting changes in the index of refraction around the nanoparticles are detected by phase-sensitive OCT. Lock-in detection of the pump beam amplitude-modulated frequency and the detector frequency allow for high-sensitivity images of molecular targets. This approach is attractive for nondestructive three-dimensional molecular imaging deep (approximately 2 mm) within biological samples. The protocols described here achieve a sensitivity of 14 parts per million (weight/weight) nanoparticles in the sample, which is sufficient to differentiate EGFR (epidermal growth factor receptor)-overexpressing cells from minimally expressing cells in three-dimensional cell constructs.
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11 MeSH Terms
Whole-cell analysis of low-density lipoprotein uptake by macrophages using STEM tomography.
Baudoin JP, Jerome WG, Kübel C, de Jonge N
(2013) PLoS One 8: e55022
MeSH Terms: Cell Line, Gold, Humans, Lipoproteins, LDL, Macrophages, Metal Nanoparticles, Microscopy, Electron, Scanning Transmission, Protein Transport
Show Abstract · Added March 20, 2014
Nanoparticles of heavy materials such as gold can be used as markers in quantitative electron microscopic studies of protein distributions in cells with nanometer spatial resolution. Studying nanoparticles within the context of cells is also relevant for nanotoxicological research. Here, we report a method to quantify the locations and the number of nanoparticles, and of clusters of nanoparticles inside whole eukaryotic cells in three dimensions using scanning transmission electron microscopy (STEM) tomography. Whole-mount fixed cellular samples were prepared, avoiding sectioning or slicing. The level of membrane staining was kept much lower than is common practice in transmission electron microscopy (TEM), such that the nanoparticles could be detected throughout the entire cellular thickness. Tilt-series were recorded with a limited tilt-range of 80° thereby preventing excessive beam broadening occurring at higher tilt angles. The 3D locations of the nanoparticles were nevertheless determined with high precision using computation. The obtained information differed from that obtained with conventional TEM tomography data since the nanoparticles were highlighted while only faint contrast was obtained on the cellular material. Similar as in fluorescence microscopy, a particular set of labels can be studied. This method was applied to study the fate of sequentially up-taken low-density lipoprotein (LDL) conjugated to gold nanoparticles in macrophages. Analysis of a 3D reconstruction revealed that newly up-taken LDL-gold was delivered to lysosomes containing previously up-taken LDL-gold thereby forming onion-like clusters.
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8 MeSH Terms
Biomimetic monolayer-protected gold nanoparticles for immunorecognition.
Harkness KM, Turner BN, Agrawal AC, Zhang Y, McLean JA, Cliffel DE
(2012) Nanoscale 4: 3843-51
MeSH Terms: Animals, Antibodies, Antigens, Biomimetic Materials, Epitopes, Gold, Metal Nanoparticles, Mice, Peptides, Tissue Distribution
Show Abstract · Added January 20, 2015
Gold nanoparticles (AuNPs) protected by self-assembled monolayers (SAMs) are capable of presenting precisely engineered surfaces at the nanoscale, allowing the mimicry of biomacromolecules on an artificial platform. Here we review the generation, characterization, and applications of monolayer-protected AuNPs that have been designed for immunorecognition by the integration of an oligopeptide epitope into the protecting monolayer. The resulting peptide-AuNP conjugate is an effective platform for biomimesis, as demonstrated by multiple studies. Recent work is presented and future directions for this field of research are discussed.
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10 MeSH Terms