163 results on '"Eunkeu Oh"'
Search Results
2. Self-assembled nanoparticle-enzyme aggregates enhance functional protein production in pure transcription-translation systems
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Meghna Thakur, Joyce C. Breger, Kimihiro Susumu, Eunkeu Oh, Joseph R. Spangler, Igor L. Medintz, Scott A. Walper, and Gregory A. Ellis
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Medicine ,Science - Abstract
Cell-free protein synthesis systems (CFPS) utilize cellular transcription and translation (TX-TL) machinery to synthesize proteins in vitro. These systems are useful for multiple applications including production of difficult proteins, as high-throughput tools for genetic circuit screening, and as systems for biosensor development. Though rapidly evolving, CFPS suffer from some disadvantages such as limited reaction rates due to longer diffusion times, significant cost per assay when using commercially sourced materials, and reduced reagent stability over prolonged periods. To address some of these challenges, we conducted a series of proof-of-concept experiments to demonstrate enhancement of CFPS productivity via nanoparticle assembly driven nanoaggregation of its constituent proteins. We combined a commercially available CFPS that utilizes purified polyhistidine-tagged (His-tag) TX-TL machinery with CdSe/CdS/ZnS core/shell/shell quantum dots (QDs) known to readily coordinate His-tagged proteins in an oriented fashion. We show that nanoparticle scaffolding of the CFPS cross-links the QDs into nanoaggregate structures while enhancing the production of functional recombinant super-folder green fluorescent protein and phosphotriesterase, an organophosphate hydrolase; the latter by up to 12-fold. This enhancement, which occurs by an undetermined mechanism, has the potential to improve CFPS in general and specifically CFPS-based biosensors (faster response time) while also enabling rapid detoxification/bioremediation through point-of-concern synthesis of similar catalytic enzymes. We further show that such nanoaggregates improve production in diluted CFPS reactions, which can help to save money and extend the amount of these costly reagents. The results are discussed in the context of what may contribute mechanistically to the enhancement and how this can be applied to other CFPS application scenarios.
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- 2022
3. A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike.
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Ying Fu, Juliana da Fonseca Rezende E Mello, Bryan D Fleming, Alex Renn, Catherine Z Chen, Xin Hu, Miao Xu, Kirill Gorshkov, Quinlin Hanson, Wei Zheng, Emily M Lee, Lalith Perera, Robert Petrovich, Manisha Pradhan, Richard T Eastman, Zina Itkin, Thomas B Stanley, Allen Hsu, Venkata Dandey, Kedar Sharma, William Gillette, Troy Taylor, Nitya Ramakrishnan, Shelley Perkins, Dominic Esposito, Eunkeu Oh, Kimihiro Susumu, Mason Wolak, Marc Ferrer, Matthew D Hall, Mario J Borgnia, and Anton Simeonov
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Medicine ,Science - Abstract
Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.
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- 2022
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4. Liquid Crystal Nanoparticle Conjugates for Scavenging Reactive Oxygen Species in Live Cells
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Okhil K. Nag, Jawad Naciri, Kwahun Lee, Eunkeu Oh, Bethany Almeida, and James B. Delehanty
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liquid crystal nanoparticles ,ROS scavenger ,oxidative stress ,TEMPO ,lipid peroxidation ,reactive oxygen species ,Medicine ,Pharmacy and materia medica ,RS1-441 - Abstract
The elevated intracellular production of or extracellular exposure to reactive oxygen species (ROS) causes oxidative stress to cells, resulting in deleterious irreversible biomolecular reactions (e.g., lipid peroxidation) and disease progression. The use of low-molecular weight antioxidants, such as 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), as ROS scavengers fails to achieve the desired efficacy because of their poor or uncontrolled cellular uptake and off-target effects, such as dysfunction of essential redox homeostasis. In this study, we fabricated a liquid crystal nanoparticle (LCNP) conjugate system with the fluorescent dye perylene (PY) loaded in the interior and poly (ethylene glycol) (PEG) decorated on the surface along with multiple molecules of TEMPO (PY-LCNP-PEG/TEMPO). PY-LCNP-PEG/TEMPO exhibit enhanced cellular uptake, and efficient ROS-scavenging activity in live cells. On average, the 120 nm diameter PY-LCNPs were conjugated with >1800 molecules of TEMPO moieties on their surface. PY-LCNP-PEG/TEMPO showed significantly greater reduction in ROS activity and lipid peroxidation compared to free TEMPO when the cells were challenged with ROS generating agents, such as hydrogen peroxide (H2O2). We suggest that this is due to the increased local concentration of TEMPO molecules on the surface of the PY-LCNP-PEG/TEMPO NPs, which efficiently bind to the plasma membrane and enter cells. Overall, these results demonstrate the enhanced capability of TEMPO-conjugated LCNPs to protect live cells from oxidative stress by effectively scavenging ROS and reducing lipid peroxidation.
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- 2022
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5. Fabrication of Photoluminescent Quantum Dot Thiol–yne Nanocomposites via Thermal Curing or Photopolymerization
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Michael H. Stewart, Kimihiro Susumu, Eunkeu Oh, Christopher G. Brown, Collin C. McClain, Edward P. Gorzkowski, and Darryl A. Boyd
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Chemistry ,QD1-999 - Published
- 2018
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6. Conjugation of biotin-coated luminescent quantum dots with single domain antibody-rhizavidin fusions
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Jinny L. Liu, Scott A. Walper, Kendrick B. Turner, Audrey Brozozog Lee, Igor L. Medintz, Kimihiro Susumu, Eunkeu Oh, Dan Zabetakis, Ellen R. Goldman, and George P. Anderson
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Single domain antibodies ,Rhizavidin ,Quantum dots ,Surface plasmon resonance ,Biotechnology ,TP248.13-248.65 - Abstract
Straightforward and effective methods are required for the bioconjugation of proteins to surfaces and particles. Previously we demonstrated that the fusion of a single domain antibody with the biotin binding molecule rhizavidin provided a facile method to coat biotin-modified surfaces with a highly active and oriented antibody. Here, we constructed similar single domain antibody—rhizavidin fusions as well as unfused rhizavidin with a His-tag. The unfused rhizavidin produced efficiently and its utility for assay development was demonstrated in surface plasmon resonance experiments. The single domain antibody-rhizavidin fusions were utilized to coat quantum dots that had been prepared with surface biotins. Preparation of antibody coated quantum dots by this means was found to be both easy and effective. The prepared single domain antibody-quantum dot reagent was characterized by surface plasmon resonance and applied to toxin detection in a fluoroimmunoassay sensing format.
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- 2016
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7. Modulation of Intracellular Quantum Dot to Fluorescent Protein Förster Resonance Energy Transfer via Customized Ligands and Spatial Control of Donor–Acceptor Assembly
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Lauren D. Field, Scott A. Walper, Kimihiro Susumu, Eunkeu Oh, Igor L. Medintz, and James B. Delehanty
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fluorescence ,sensor ,FRET ,quantum dot ,protein ,membrane ,assembly ,noncovalent ,ligand ,Chemical technology ,TP1-1185 - Abstract
Understanding how to controllably modulate the efficiency of energy transfer in Förster resonance energy transfer (FRET)-based assemblies is critical to their implementation as sensing modalities. This is particularly true for sensing assemblies that are to be used as the basis for real time intracellular sensing of intracellular processes and events. We use a quantum dot (QD) donor -mCherry acceptor platform that is engineered to self-assemble in situ wherein the protein acceptor is expressed via transient transfection and the QD donor is microinjected into the cell. QD-protein assembly is driven by metal-affinity interactions where a terminal polyhistidine tag on the protein binds to the QD surface. Using this system, we show the ability to modulate the efficiency of the donor–acceptor energy transfer process by controllably altering either the ligand coating on the QD surface or the precise location where the QD-protein assembly process occurs. Intracellularly, a short, zwitterionic ligand mediates more efficient FRET relative to longer ligand species that are based on the solubilizing polymer, poly(ethylene glycol). We further show that a greater FRET efficiency is achieved when the QD-protein assembly occurs free in the cytosol compared to when the mCherry acceptor is expressed tethered to the inner leaflet of the plasma membrane. In the latter case, the lower FRET efficiency is likely attributable to a lower expression level of the mCherry acceptor at the membrane combined with steric hindrance. Our work points to some of the design considerations that one must be mindful of when developing FRET-based sensing schemes for use in intracellular sensing.
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- 2015
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8. Photothermal-Enhanced Modulation of Cellular Membrane Potential Using Long-Wavelength-Activated Gold Nanoflowers
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Katherine E. Rogers, Okhil K. Nag, Kimihiro Susumu, Eunkeu Oh, and James B. Delehanty
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Pharmacology ,Organic Chemistry ,Biomedical Engineering ,Pharmaceutical Science ,Bioengineering ,Biotechnology - Published
- 2023
9. Seedless Synthesis of Disulfide-Grafted Gold Nanoflowers with Size and Shape Control and Their Photothermally Mediated Cell Perforation
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Ajmeeta Sangtani, Kwahun Lee, Okhil K. Nag, Kimihiro Susumu, R. Joseph Weiblen, Mijin Kim, Igor Vurgaftman, Sz-Chian Liou, James B. Delehanty, and Eunkeu Oh
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General Chemical Engineering ,Materials Chemistry ,General Chemistry - Published
- 2022
10. Benzaldehyde Lyase Kinetic Improvements, Potential Channeling to Alcohol Dehydrogenase, and Substrate Scope when Immobilized on Semiconductor Quantum Dots
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Shelby Hooe, Joyce Breger, Scott Dean, Kimihiro Susumu, Eunkeu Oh, Scott Walper, Gregory A. Ellis, and Igor L. Medintz
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General Materials Science - Published
- 2022
11. Mechanistic Understanding of DNA Denaturation in Nanoscale Thermal Gradients Created by Femtosecond Excitation of Gold Nanoparticles
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David A. Hastman, Parth Chaturvedi, Eunkeu Oh, Joseph S. Melinger, Igor L. Medintz, Lela Vuković, and Sebastián A. Díaz
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Time Factors ,Materials Testing ,Temperature ,Metal Nanoparticles ,Biocompatible Materials ,General Materials Science ,DNA ,Gold ,Molecular Dynamics Simulation ,Nucleic Acid Denaturation - Abstract
There is significant interest in developing photothermal systems that can precisely control the structure and function of biomolecules through local temperature modulation. One specific application is the denaturation of double-stranded (ds) DNA through femtosecond (fs) laser pulse optical heating of gold nanoparticles (AuNPs); however, the mechanism of DNA melting in these systems is not fully understood. Here, we utilize 55 nm AuNPs with surface-tethered dsDNA, which are locally heated using fs laser pulses to induce DNA melting. By varying the dsDNA distance from the AuNP surface and the laser pulse energy fluence, this system is used to study how the nanosecond duration temperature increase and the steep temperature gradient around the AuNP affect dsDNA dehybridization. Through modifying the distance between the dsDNA and AuNP surface by 3.8 nm in total and the pulse energy fluence from 7.1 to 14.1 J/m
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- 2022
12. Exploring the performance of Escherichia coli outer membrane vesicles as a tool for vaccine development against Chagas disease
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María Elisa Vázquez, Andrea Cecilia Mesías, Leonardo Acuña, Joseph Spangler, Brenda Zabala, Cecilia Parodi, Meghna Thakur, Eunkeu Oh, Scott Allan Walper, and Cecilia Pérez Brandán
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Microbiology (medical) ,Trypanosoma cruzi ,vaccine ,outer membrane vesicle - Abstract
BACKGROUND Vaccine development is a laborious craftwork in which at least two main components must be defined: a highly immunogenic antigen and a suitable delivery method. Hence, the interplay of these elements could elicit the required immune response to cope with the targeted pathogen with a long-lasting protective capacity. OBJECTIVES Here we evaluate the properties of Escherichia coli spherical proteoliposomes - known as outer membrane vesicles (OMVs) - as particles with natural adjuvant capacities and as antigen-carrier structures to assemble an innovative prophylactic vaccine for Chagas disease. METHODS To achieve this, genetic manipulation was carried out on E. coli using an engineered plasmid containing the Tc24 Trypanosoma cruzi antigen. The goal was to induce the release of OMVs displaying the parasite protein on their surface. FINDINGS As a proof of principle, we observed that native OMVs - as well as those carrying the T. cruzi antigen - were able to trigger a slight, but functional humoral response at low immunization doses. Of note, compared to the non-immunized group, native OMVs-vaccinated animals survived the lethal challenge and showed minor parasitemia values, suggesting a possible involvement of innate trained immunity mechanism. MAIN CONCLUSION These results open the range for further research on the design of new carrier strategies focused on innate immunity activation as an additional immunization target and venture to seek for alternative forms in which OMVs could be used for optimizing vaccine development.
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- 2023
13. Enzyme Assembly on Nanoparticle Scaffolds Enhances Cofactor Recycling and Improves Coupled Reaction Kinetics
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Joyce C. Breger, Ellen R Goldman, Kimihiro Susumu, Eunkeu Oh, Christopher M Green, Shelby Hooe, Meghna Thakur, Igor Medintz, and Gregory A. Ellis
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General Materials Science - Abstract
Enzyme activity can be many times enhanced in configurations where they are displayed on a nanoparticle (NP) and this same format sometimes even provides access to channeling phenomena within multienzyme...
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- 2023
14. Fluorescent quantum dots enable SARS-CoV-2 antiviral drug discovery and development
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Kirill Gorshkov, Mason A. Wolak, Eunkeu Oh, and Kimihiro Susumu
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SARS-CoV-2 ,medicine.drug_class ,viruses ,Computational biology ,Biology ,medicine.disease_cause ,Antiviral Agents ,Small molecule ,Virus ,COVID-19 Drug Treatment ,Drug repositioning ,Immune system ,Quantum dot ,Biosafety level ,Quantum Dots ,Drug Discovery ,medicine ,Humans ,Antiviral drug ,Coronavirus - Abstract
Introduction SARS-CoV-2 is a highly infectious and deadly coronavirus whose study requires the use of a biosafety level 3 (BSL-3) containment facility to investigate viral biology and pathogenesis, which limits the study of live virus and slows progress towards finding suitable treatments for infection. While vaccines from several companies have proven very effective in combating the virus, few treatments exist for those who do succumb to the viral-induced systemic disease called COVID-19. Areas covered This short review focuses on fluorescent quantum dot-based modeling of SARS-CoV-2. New BSL-2 viral models are essential to find small molecules and biologics that may be effective in stopping viral infection as well as treating already infected individuals. Nanoparticles are invaluable tools for biological research as they can be used to both modeling pathogens and serve as a platform for developing vaccines. Expert opinion Visualizing viral activity with fluorescent quantum dots enables both biochemical and cell-based assays to detect virus-host receptor interactions, cellular activity after binding to cell plasma membrane, screening for interventions using small molecule drug repurposing, and testing of novel biologics. Quantum dots can also be used for diagnostic assays, vaccine development, and importantly, pan-antiviral drugs to address variants that may escape the immune response.
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- 2021
15. Hybrid Nucleic Acid-Quantum Dot Assemblies as Multiplexed Reporter Platforms for Cell-Free Transcription Translation-Based Biosensors
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Divita Mathur, Meghna Thakur, Sebastián A. Díaz, Kimihiro Susumu, Michael H. Stewart, Eunkeu Oh, Scott A. Walper, and Igor L. Medintz
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Biomedical Engineering ,General Medicine ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Article - Abstract
Cell-free synthetic biology has emerged as a valuable tool for the development of rapid, portable biosensors that can be readily transported in the freeze-dried form to the point of need eliminating cold chain requirements. One of the challenges associated with cell-free sensors is the ability to simultaneously detect multiple analytes within a single reaction due to the availability of a limited set of fluorescent and colorimetric reporters. To potentially provide multiplexing capabilities to cell-free biosensors, we designed a modular semiconductor quantum dot (QD)-based reporter platform that is plugged in downstream of the transcription-translation functionality in the cell-free reaction and which converts enzymatic activity in the reaction into distinct optical signals. We demonstrate proof of concept by converting restriction enzyme activity, utilized as our prototypical sensing output, into optical changes across several distinct spectral output channels that all use a common excitation wavelength. These hybrid Förster resonance energy transfer (FRET)-based QD peptide PNA-DNA-Dye reporters (QD-PDDs) are completely self-assembled and consist of differentially emissive QD donors paired to a dye-acceptor displayed on a unique DNA encoding a given enzyme’s cleavage site. Three QD-based PDDs, independently activated by the enzymes BamHI, EcoRI, and NcoI, were prototyped in mixed enzyme assays where all three demonstrated the ability to convert enzymatic activity into fluorescent output. Simultaneous monitoring of each of the three paired QD-donor dye-acceptor spectral channels in cell-free biosensing reactions supplemented with added linear genes encoding each enzyme confirmed robust multiplexing capabilities for at least two enzymes when co-expressed. The modular QD-PDDs are easily adapted to respond to other restriction enzymes or even proteases if desired.
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- 2022
16. Microbial survival and growth on <scp>non‐corrodible</scp> conductive materials
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Leonard M. Tender, Sarah M. Glaven, Eunkeu Oh, Brian J. Eddie, Daniel A. Phillips, and Lina J. Bird
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Microbial fuel cell ,Bioelectric Energy Sources ,Open-circuit voltage ,Carbon fixation ,Electric Conductivity ,Biofilm ,Biology ,Chromatiaceae ,Microbiology ,Redox ,Corrosion ,Chemical engineering ,Biofilms ,Electrode ,Graphite ,Electrodes ,Oxidation-Reduction ,Ecology, Evolution, Behavior and Systematics - Abstract
Biofilms growing aerobically on conductive substrates are often correlated with a positive, sustained shift in their redox potential. This phenomenon has a beneficial impact on microbial fuel cells by increasing their overall power output but can be detrimental when occurring on stainless steel by enhancing corrosion. The biological mechanism behind this potential shift is unresolved and a metabolic benefit to cells has not been demonstrated. Here, biofilms containing the electroautotroph 'Candidatus Tenderia electrophaga' catalysed a shift in the open circuit potential of graphite and indium tin oxide electrodes by >100 mV. Biofilms on open circuit electrodes had increased biomass and a significantly higher proportion of 'Ca. Tenderia electrophaga' compared to those on plain glass. Addition of metabolic inhibitors showed that living cells were required to maintain the more positive potential. We propose a model to describe these observations, in which 'Ca. Tenderia electrophaga' drives the shift in open circuit potential through electron uptake for oxygen reduction and CO2 fixation. We further propose that the electrode is continuously recharged by oxidation of trace redox-active molecules in the medium at the more positive potential. A similar phenomenon is possible on natural conductive substrates in the environment.
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- 2021
17. Excited-State Dynamics of Photoluminescent Gold Nanoclusters and Their Assemblies with Quantum Dot Donors
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Igor L. Medintz, Eunkeu Oh, Shiori Yamazaki, Amy M. Scott, and Kimihiro Susumu
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General Energy ,Photoluminescence ,Materials science ,Quantum dot ,Excited state ,Dynamics (mechanics) ,Physical and Theoretical Chemistry ,Molecular physics ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Nanoclusters - Published
- 2021
18. Direct and Efficient Conjugation of Quantum Dots to DNA Nanostructures with Peptide-PNA
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Divita Mathur, Eunkeu Oh, Igor L. Medintz, Sebastián A. Díaz, Kimihiro Susumu, David A. Hastman, and Christopher Green
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Peptide Nucleic Acids ,General Physics and Astronomy ,Nanoparticle ,Nanotechnology ,02 engineering and technology ,Conjugated system ,010402 general chemistry ,01 natural sciences ,chemistry.chemical_compound ,Quantum Dots ,DNA nanotechnology ,General Materials Science ,Bioconjugation ,Chemistry ,technology, industry, and agriculture ,General Engineering ,DNA ,021001 nanoscience & nanotechnology ,Nanostructures ,0104 chemical sciences ,Quantum dot ,Nucleic acid ,Peptides ,0210 nano-technology ,Conjugate - Abstract
DNA nanotechnology has proven to be a powerful strategy for the bottom-up preparation of colloidal nanoparticle (NP) superstructures, enabling the coordination of multiple NPs with orientation and separation approaching nanometer precision. To do this, NPs are often conjugated with chemically modified, single-stranded (ss) DNA that can recognize complementary ssDNA on the DNA nanostructure. The limitation is that many NPs cannot be easily conjugated with ssDNA, and other conjugation strategies are expensive, inefficient, or reduce the specificity and/or precision with which NPs can be placed. As an alternative, the conjugation of nanoparticle-binding peptides and peptide nucleic acids (PNA) can produce peptide-PNA with distinct NP-binding and DNA-binding domains. Here, we demonstrate a simple application of this method to conjugate semiconductor quantum dots (QDs) directly to DNA nanostructures by means of a peptide-PNA with a six-histidine peptide motif that binds to the QD surface. With this method, we achieved greater than 90% capture efficiency for multiple QDs on a single DNA nanostructure while preserving both site specificity and precise spatial control of QD placement. Additionally, we investigated the effects of peptide-PNA charge on the efficacy of QD immobilization in suboptimal conditions. The results validate peptide-PNA as a viable alternative to ssDNA conjugation of NPs and warrant studies of other NP-binding peptides for peptide-PNA conjugation.
- Published
- 2021
19. Polyhistidine-Tag-Enabled Conjugation of Quantum Dots and Enzymes to DNA Nanostructures
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Christopher M, Green, Divita, Mathur, Kimihiro, Susumu, Eunkeu, Oh, Igor L, Medintz, and Sebastián A, Díaz
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Quantum Dots ,Histidine ,DNA ,Coloring Agents ,Luciferases ,Nanostructures - Abstract
DNA nanostructures self-assemble into almost any arbitrary architecture, and when combined with their capability to precisely position and orient dyes, nanoparticles, and biological moieties, the technology reaches its potential. We present a simple yet multifaceted conjugation strategy based on metal coordination by a multi-histidine peptide tag (Histag). The versatility of the Histag as a means to conjugate to DNA nanostructures is shown by using Histags to capture semiconductor quantum dots (QDs) with numerical and positional precision onto a DNA origami breadboard. Additionally, Histag-expressing enzymes, such as the bioluminescent luciferase, can also be captured to the DNA origami breadboard with similar precision. DNA nanostructure conjugation of the QDs or luciferase is confirmed through imaging and/or energy transfer to organic dyes integrated into the DNA nanostructure.
- Published
- 2022
20. Gold Nanoparticle Templating Increases the Catalytic Rate of an Amylase, Maltase, and Glucokinase Multienzyme Cascade through Substrate Channeling Independent of Surface Curvature
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Priscilla Choo, Scott A. Walper, Igor L. Medintz, Kimihiro Susumu, William P. Klein, David A. Hastman, Eunkeu Oh, Sebastián A. Díaz, and Teri W. Odom
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biology ,Glucokinase ,Chemistry ,education ,Substrate channeling ,Nanoparticle ,General Chemistry ,Catalysis ,Enzyme catalysis ,Colloidal gold ,biology.protein ,Biophysics ,Amylase ,Maltase ,health care economics and organizations - Abstract
The templating of enzymes has shown myriad advantages, including increased stability and kinetic rates. Specifically, the use of nanoparticles (NPs) as templates has been shown to increase the kine...
- Published
- 2020
21. High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells
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Kirill Gorshkov, Mason Wolak, Kimihiro Susumu, Eunkeu Oh, and Bruce Nguyen Tran
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HEK293 Cells ,General Immunology and Microbiology ,SARS-CoV-2 ,General Chemical Engineering ,General Neuroscience ,Quantum Dots ,Spike Glycoprotein, Coronavirus ,Humans ,Endocytosis ,General Biochemistry, Genetics and Molecular Biology - Abstract
The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum dots are fluorescent nanoparticles with excellent photophysical properties imbued with bright and stable photoluminescence as well as narrow emission bands. Quantum dots are spherical in shape, and with the proper modification of the surface chemistry, can be used to conjugate biomolecules for cellular applications. These optical properties, combined with the ability to functionalize them with biomolecules, make them an excellent tool for investigating receptor-ligand interactions and cellular trafficking. Here, we present a method that uses quantum dots to track the binding and endocytosis of SARS-CoV-2 spike protein. This protocol can be used as a guide for experimentalists looking to utilize quantum dots to study protein-protein interactions and trafficking in the context of cellular physiology.
- Published
- 2022
22. Femtosecond Laser Pulse Excitation of DNA-Labeled Gold Nanoparticles: Establishing a Quantitative Local Nanothermometer for Biological Applications
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Matthew Chiriboga, Eunkeu Oh, Joseph S. Melinger, Sebastián A. Díaz, Carl W. Brown, David A. Hastman, Paul D. Cunningham, Igor L. Medintz, Zachary J. Salvato, Thomas M. Salvato, Guillermo Lasarte Aragonés, and Divita Mathur
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Materials science ,Metal Nanoparticles ,General Physics and Astronomy ,Nanoparticle ,02 engineering and technology ,010402 general chemistry ,01 natural sciences ,law.invention ,Nucleic acid thermodynamics ,law ,General Materials Science ,Plasmonic nanoparticles ,Lasers ,Photothermal effect ,Temperature ,General Engineering ,DNA ,Photothermal therapy ,021001 nanoscience & nanotechnology ,Laser ,0104 chemical sciences ,Chemical physics ,Colloidal gold ,Femtosecond ,Gold ,0210 nano-technology - Abstract
Femtosecond (fs) laser pulsed excitation of plasmonic nanoparticle (NP)-biomolecule conjugates is a promising method to locally heat biological materials. Studies have demonstrated that fs pulses of light can modulate the activity of DNA or proteins when attached to plasmonic NPs; however, the precision over subsequent biological function remains largely undetermined. Specifically, the temperature the localized biomolecules "experience" remains unknown. We used 55 nm gold nanoparticles (AuNPs) displaying double-stranded (ds) DNA to examine how, for dsDNA with different melting temperatures, the laser pulse energy fluence and bulk solution temperature affect the rate of local DNA denaturation. A universal "template" single-stranded DNA was attached to the AuNP surface, and three dye-labeled probe strands, distinct in length and melting temperature, were hybridized to it creating three individual dsDNA-AuNP bioconjugates. The dye-labeled probe strands were used to quantify the rate and amount of DNA release after a given number of light pulses, which was then correlated to the dsDNA denaturation temperature, resulting in a quantitative nanothermometer. The localized DNA denaturation rate could be modulated by more than threefold over the biologically relevant range of 8-53 °C by varying pulse energy fluence, DNA melting temperature, and surrounding bath temperature. With a modified dissociation equation tailored for this system, a "sensed" temperature parameter was extracted and compared to simulated AuNP temperature profiles. Determining actual biological responses in such systems can allow researchers to design precision nanoscale photothermal heating sources.
- Published
- 2020
23. Anionic Conjugated Polyelectrolytes for FRET‐based Imaging of Cellular Membrane Potential
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Eunkeu Oh, Han Young Woo, James B. Delehanty, Van Sang Le, Okhil K. Nag, and Ji Eun Jeong
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Anions ,Patch-Clamp Techniques ,Fluorophore ,02 engineering and technology ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Membrane Potentials ,chemistry.chemical_compound ,Fluorescence Resonance Energy Transfer ,Humans ,Physical and Theoretical Chemistry ,Fluorescent Dyes ,Membrane potential ,Cell Membrane ,Depolarization ,General Medicine ,021001 nanoscience & nanotechnology ,Polyelectrolytes ,Acceptor ,Fluorescence ,Conjugated Polyelectrolytes ,0104 chemical sciences ,HEK293 Cells ,Membrane ,Förster resonance energy transfer ,chemistry ,Biophysics ,0210 nano-technology - Abstract
We report a Forster resonance energy transfer (FRET)-based imaging ensemble for the visualization of membrane potential in living cells. A water-soluble poly(fluorene-cophenylene) conjugated polyelectrolyte (FsPFc10) serves as a FRET donor to a voltage-sensitive dye acceptor (FluoVolt™ ). We observe FRET between FsPFc10 and FluoVolt™ , where the enhancement in FRET-sensitized emission from FluoVolt™ is measured at various donor/acceptor ratios. At a donor/acceptor ratio of 1, the excitation of FluoVolt™ in a FRET configuration results in a three-fold enhancement in its fluorescence emission (compared to when it is excited directly). FsPFc10 efficiently labels the plasma membrane of HEK 293T/17 cells and remains resident with minimal cellular internalization for ~ 1.5 h. The successful plasma membrane-associated colabeling of the cells with the FsPFc10-FluoVolt™ donor-acceptor pair is confirmed by dual-channel confocal imaging. Importantly, cells labeled with FsPFc10 show excellent cellular viability with no adverse effect on cell membrane depolarization. During depolarization of membrane potential, HEK 293T/17 cells labeled with the donor-acceptor FRET pair exhibit a greater fluorescence response in FluoVolt™ emission relative to when FluoVolt™ is used as the sole imaging probe. These results demonstrate the conjugated polyelectrolyte to be a new class of membrane labeling fluorophore for use in voltage sensing schemes.
- Published
- 2020
24. Gold-Nanoparticle-Mediated Depolarization of Membrane Potential Is Dependent on Concentration and Tethering Distance from the Plasma Membrane
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Megan E. Muroski, Igor L. Medintz, Eunkeu Oh, Okhil K. Nag, Alan L. Huston, James B. Delehanty, and Alexander L. Efros
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Biomedical Engineering ,Metal Nanoparticles ,Pharmaceutical Science ,Nanoparticle ,Bioengineering ,02 engineering and technology ,PC12 Cells ,01 natural sciences ,Membrane Potentials ,Polyethylene Glycols ,chemistry.chemical_compound ,PEG ratio ,Animals ,Pharmacology ,Membrane potential ,Dose-Response Relationship, Drug ,010405 organic chemistry ,Cell Membrane ,Organic Chemistry ,Depolarization ,Photothermal therapy ,021001 nanoscience & nanotechnology ,Rats ,0104 chemical sciences ,Cholesterol ,Membrane ,chemistry ,Colloidal gold ,Biophysics ,Gold ,0210 nano-technology ,Ethylene glycol ,Biotechnology - Abstract
The photoactivation of plasma-membrane-tethered gold nanoparticles (AuNPs) for the photothermally driven depolarization of membrane potential has recently emerged as a new platform for the controlled actuation of electrically active cells. In this report, we characterize the relationship between AuNP concentration and AuNP-membrane separation distance with the efficiency of photoactivated plasma membrane depolarization. We show in differentiated rat pheochromocytoma (PC-12) cells that AuNPs capped with poly(ethylene glycol) (PEG)-cholesterol ligands localize to the plasma membrane and remain resident for up to 1 h. The efficiency of AuNP-mediated depolarization is directly dependent on the concentration of the NPs on the cell surface. We further show that the efficiency of AuNP-mediated photothermal depolarization of membrane potential is directly dependent on the tethering distance between the AuNP and the plasma membrane, which we control by iteratively tuning the length of the PEG linker. Importantly, the AuNP conjugates do not adversely affect cell viability under the photoactivation conditions required for membrane depolarization. Our results demonstrate the fine control that can be elicited over AuNP bioconjugates and establishes principles for the rational design of functional nanomaterials for the control of electrically excitable cells.
- Published
- 2020
25. Polyhistidine-Tag-Enabled Conjugation of Quantum Dots and Enzymes to DNA Nanostructures
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Christopher M. Green, Divita Mathur, Kimihiro Susumu, Eunkeu Oh, Igor L. Medintz, and Sebastián A. Díaz
- Published
- 2022
26. A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike
- Author
-
Ying Fu, Juliana da Fonseca Rezende e Mello, Bryan D. Fleming, Alex Renn, Catherine Z. Chen, Xin Hu, Miao Xu, Kirill Gorshkov, Quinlin Hanson, Wei Zheng, Emily M. Lee, Lalith Perera, Robert Petrovich, Manisha Pradhan, Richard T. Eastman, Zina Itkin, Thomas B. Stanley, Allen Hsu, Venkata Dandey, Kedar Sharma, William Gillette, Troy Taylor, Nitya Ramakrishnan, Shelley Perkins, Dominic Esposito, Eunkeu Oh, Kimihiro Susumu, Mason Wolak, Marc Ferrer, Matthew D. Hall, Mario J. Borgnia, and Anton Simeonov
- Subjects
Mutation ,Multidisciplinary ,biology ,SARS-CoV-2 ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,COVID-19 ,Spike Protein ,Human airway ,Single-Domain Antibodies ,Antibodies, Viral ,medicine.disease_cause ,Antibodies, Neutralizing ,Virology ,Article ,Neutralization ,Spike Glycoprotein, Coronavirus ,medicine ,biology.protein ,Humans ,Bacteriophages ,Spike (software development) ,Antibody ,Ex vivo ,Protein Binding - Abstract
Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants. ONE-SENTENCE SUMMARY: A cost-effective, high-throughput, adaptable pipeline capable of identifying effective humanized nanobodies against SARS-CoV-2.
- Published
- 2021
27. Quantum dot-enabled membrane-tethering and enhanced photoactivation of chlorin-e6
- Author
-
Ajmeeta Sangtani, Michael H. Stewart, James B. Delehanty, Okhil K. Nag, and Eunkeu Oh
- Subjects
Materials science ,Membrane permeability ,medicine.medical_treatment ,Bioengineering ,Photodynamic therapy ,Context (language use) ,General Chemistry ,Condensed Matter Physics ,Atomic and Molecular Physics, and Optics ,Förster resonance energy transfer ,Membrane ,Quantum dot ,Modeling and Simulation ,medicine ,Biophysics ,General Materials Science ,Photosensitizer ,Conjugate - Abstract
Photodynamic therapy (PDT) has emerged as an attractive therapeutic modality for the targeted destruction of abnormal cells as it involves the specific generation of reactive oxygen species (ROS) in tissue only in the combined presence of a photosensitizer (PS), incident excitation light, and molecular oxygen. A variety of effective PS molecules have been developed but they are often limited by poor water solubility or a lack of cell-type specificity. We have developed a quantum dot-chlorin e6 (QD-Ce6) nanobioconjugate system where the QD (5 nm diameter) serves simultaneously as a hydrophilic scaffold and an efficient Forster resonance energy transfer (FRET) donor to multiple Ce6 PS acceptors arrayed around the central QD. Decoration of the conjugate with a membrane-tethering peptide stably localizes the ensemble conjugate system on the exofacial leaflet of the plasma membrane of mammalian cells. Excitation of Ce6 in a FRET configuration results in membrane-localized ROS generation resulting in lipid peroxidation, increased membrane permeability, and inhibition of cellular proliferation. We present and discuss our results in the context of the further evolution of QD-based PDT systems.
- Published
- 2021
28. Gold nanoparticles capable of templating entire enzyme cascades and improving production yield through substrate channeling
- Author
-
Sebastián A. Díaz, David A. Hastman, Eunkeu Oh, Scott A. Walper, and Igor L. Medintz
- Subjects
chemistry.chemical_classification ,Enzyme ,Biocatalysis ,Chemistry ,Colloidal gold ,education ,Substrate channeling ,Kinetics ,technology, industry, and agriculture ,Biophysics ,Nanoparticle ,Maltase ,Conjugate - Abstract
We have demonstrated that multi-enzyme cascades can be templated on individual gold nanoparticles (NPs) with diameters below 100 nm. Utilizing a three enzyme cascade of amylase, maltase, and glucokinase we found a ~3-fold enhancement in product formation when all three enzymes were bound to the same NP as compared to controls. This strongly suggests that the increased kinetics was due to substrate channeling. Additional controls were realized to ensure that only when the enzymes were bound to the NPs was enhancement observed by modifying the ratio of enzyme to NP. Furthermore the experiments support a model where a single-layer of enzymes conjugate to the NPs independently of the enzyme to NP ratio. Being able to conjugate entire cascades on individual NPs should allow for optimized design of NPs and enzyme cascades for in vitro biocatalysis.
- Published
- 2021
29. Aqueous-phase N-dealkylation of fentanyl using a water-soluble rhodium(III) porphyrin complex
- Author
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Hemant Pal, Kathy Maiello, Eunkeu Oh, Okhil Nag, James Delehanty, and Anneli Nina
- Published
- 2021
30. Quantum Dot‐Conjugated SARS‐CoV‐2 Spike Nanoparticles for SARS‐CoV‐2 infection modeling and drug discovery
- Author
-
Jiji Chen, Joyce C. Breger, Manisha Pradhan, Eunkeu Oh, Miao Xu, Kirill Gorshkov, Wei Zhu, Xin Hu, Mason A. Wolak, and Kimihiro Susumu
- Subjects
Pharmacology ,Drug discovery ,Chemistry ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,Conjugated system ,Biochemistry ,Virology ,Quantum dot ,Genetics ,Spike (software development) ,Molecular Biology ,hormones, hormone substitutes, and hormone antagonists ,Pharmacology ‐ COVID‐19 ,Biotechnology - Abstract
The SARS‐CoV‐2 virus binds to host cell surface ACE2 on the plasma membrane via the spike protein's receptor binding domain. Our work has resulted in the generation of a versatile imaging probe using recombinant Spike receptor binding domain conjugated to fluorescent quantum dots (QDs). This probe is capable of engaging in energy transfer quenching with ACE2‐conjugated gold nanoparticles enabling biochemical monitoring of binding. Neutralizing antibodies and recombinant human ACE2 blocked quenching, demonstrating a specific binding interaction. In cell‐based assays, we observed immediate binding of the probe on the cell surface of ACE2‐expressing cells followed by endocytosis. Neutralizing antibodies and ACE2‐Fc fully prevented binding and endocytosis with low nanomolar potency. Importantly, we can use this QD nanoparticle probe to identify and validate inhibitors of the SARS‐CoV‐2 Spike and ACE2 receptor binding in human cells. This work enables facile, rapid, and high‐throughput biochemical‐ and cell‐based screening of inhibitors for coronavirus Spike‐mediated cell recognition and entry.
- Published
- 2021
31. Inhibiting SARS‐CoV‐2 infection with lysosomal alkalizers
- Author
-
Donald C. Lo, Catherine Chen, Kirill Gorshkov, Mason A. Wolak, Zina Itkin, Carleen Klumpp-Thomas, Eunkeu Oh, Min Shen, Robert Bostwick, Khalida Shamim, Kimihiro Susumu, Anton Simeonov, Samuel G. Michael, Bruce Nguyen Tran, Wei Zhu, Wenwei Huang, Yu-Shan Cheng, Mark J. Henderson, Wei Zheng, Lynn Rasmussen, Paul Shinn, Matthew Hall, Xin Hu, Juan Carlos de la Torre, Manisha Pradhan, and Miao Xu
- Subjects
Pharmacology ,business.industry ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,Genetics ,Medicine ,business ,Molecular Biology ,Biochemistry ,Virology ,Pharmacology ‐ COVID‐19 ,Biotechnology - Abstract
Understanding the SARS‐CoV‐2 virus’ routes of infection, virus–host–protein interactions, and mechanisms of virus‐induced cytopathic effects will greatly aid in the discovery and design of new therapeutics to treat COVID‐19. Chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID‐19, have multiple cellular effects including alkalizing lysosomes and blocking autophagy as well as exhibiting dose‐limiting toxicities in patients. To identify an alternative lysosome‐based drug repurposing opportunity we evaluated additional lysosomotropic compounds . We found that six of these compounds blocked the cytopathic effect of SARS‐CoV‐2 in Vero E6 cells with half‐maximal effective concentration (EC50) values ranging from 2.0 to 13 μM and selectivity indices (SIs; SI = CC50/EC50) ranging from 1.5‐ to >10‐fold. We demonstrate how the compounds (1) blocked lysosome functioning and autophagy, (2) prevented pseudotyped particle entry, (3) increased lysosomal pH, and (4) that ROC‐325 reduced viral titers in the EpiAirway 3D tissue model. Consistent with these findings, the siRNA knockdown of ATP6V0D1 blocked the HCoV‐NL63 cytopathic effect in LLC‐MK2 cells. Moreover, an analysis of SARS‐CoV‐2 infected Vero E6 cell lysate revealed significant dysregulation of autophagy and lysosomal function, suggesting a contribution of the lysosome to the life cycle of SARS‐CoV‐2. Our findings support targeting the lysosome to combat SARS‐CoV‐2 infections and inhibitors of lysosomal function could become an important component of drug combination therapies aimed at improving treatment and outcomes for COVID‐19.
- Published
- 2021
32. Nanoparticle–Peptide–Drug Bioconjugates for Unassisted Defeat of Multidrug Resistance in a Model Cancer Cell Line
- Author
-
Eleonora Petryayeva, W. Russ Algar, Eunkeu Oh, Guillermo Lasarte-Aragonés, Kimihiro Susumu, Ajmeeta Sangtani, Igor L. Medintz, James B. Delehanty, and Alan L. Huston
- Subjects
Drug ,Endosome ,media_common.quotation_subject ,Endocytic cycle ,Biomedical Engineering ,Pharmaceutical Science ,Antineoplastic Agents ,Bioengineering ,Nanoconjugates ,02 engineering and technology ,Drug resistance ,01 natural sciences ,Cell Line, Tumor ,Neoplasms ,Humans ,Internalization ,media_common ,Pharmacology ,Drug Carriers ,010405 organic chemistry ,Chemistry ,Organic Chemistry ,021001 nanoscience & nanotechnology ,Drug Resistance, Multiple ,0104 chemical sciences ,3. Good health ,Multiple drug resistance ,Cell killing ,Doxorubicin ,Drug Resistance, Neoplasm ,Cancer cell ,Cancer research ,Peptides ,0210 nano-technology ,Biotechnology - Abstract
Multidrug resistance (MDR) is a significant challenge in the treatment of many types of cancers as membrane-associated transporters actively pump drugs out of the cell, limiting therapeutic efficacy. While nanoparticle (NP)-based therapeutics have emerged as a mechanism for overcoming MDR, they often rely on the delivery of multiple anticancer drugs, nucleic acid hybrids, or MDR pump inhibitors. The effectiveness of these strategies, however, can be limited by their off-target toxicity or the need for genetic transfection. In this paper, we describe a NP-peptide-drug bioconjugate that achieves significant cell killing in MDR-positive cancer cells without the need for additional drugs. We use a quantum dot (QD) as a central scaffold to append two species of peptide, a cell-uptake peptide to facilitate endocytic internalization and a peptide-drug conjugate that is susceptible to cleavage by esterases found within the endocytic pathway. This approach relies on spatiotemporal control over drug release, where endosomes traffic drug away from membrane-resident pumps and release it closer to the nucleus. Cellular internalization studies showed high uptake of the NP-drug complex and nuclear localization of the drug after 48 h in MDR-positive cells. Additionally, cellular proliferation assays demonstrated a 40% decrease in cell viability for the NP-drug bioconjugate compared to free drug, confirming the utility of this system in overcoming MDR in cancer cells.
- Published
- 2019
33. Analyzing fidelity and reproducibility of DNA templated plasmonic nanostructures
- Author
-
William P. Klein, Eunkeu Oh, Divita Mathur, Paul Johns, Jawad Naciri, Igor L. Medintz, Rafaela Nita, Sebastián A. Díaz, Hieu Bui, Matthew Chiriboga, and Jake Fontana
- Subjects
Plasmonic nanoparticles ,Nanostructure ,Materials science ,Metal Nanoparticles ,Nanoparticle ,Nanotechnology ,DNA ,02 engineering and technology ,Microscopy, Atomic Force ,010402 general chemistry ,021001 nanoscience & nanotechnology ,01 natural sciences ,Nanostructures ,0104 chemical sciences ,Nanomaterials ,Template ,Microscopy, Electron, Transmission ,Spectrophotometry ,DNA origami ,General Materials Science ,Nanorod ,Gold ,0210 nano-technology ,Plasmon - Abstract
Synthetic DNA templated nanostructures offer an excellent platform for the precise spatial and orientational positioning of organic and inorganic nanomaterials. Previous reports have shown its applicability in the organization of plasmonic nanoparticles in a number of geometries for the purpose of realizing tunable nanoscale optical devices. However, translation of nanoparticle-DNA constructs to application requires additional efforts to increase scalability, reproducibility, and formation yields. Understanding all these factors is, in turn, predicated on in-depth analysis of each structure and comparing how formation changes with complexity. Towards the latter goal, we assemble seven unique plasmonic nanostructure symmetries of increasing complexity based on assembly of gold nanorods and nanoparticles on two different DNA origami templates, a DNA triangle and rhombus, and characterize them using gel electrophoresis, atomic force- and transmission electron microscopy, as well as optical spectroscopy. In particular, we focus on how much control can be elicited over yield, reproducibility, shape, size, inter-particle angles, gaps, and plasmon shifts as compared to expectations from computer simulations as structural complexity increases. We discuss how these results can contribute to establishing process principles for creating DNA templated plasmonic nanostructures.
- Published
- 2019
34. Isolation and characterization of Lactobacillus-derived membrane vesicles
- Author
-
Scott A. Walper, Eunkeu Oh, Scott N. Dean, Dagmar H. Leary, and Claretta J. Sullivan
- Subjects
0301 basic medicine ,Limosilactobacillus reuteri ,lcsh:Medicine ,Proteomics ,Article ,Biological pathway ,Cell membrane ,03 medical and health sciences ,0302 clinical medicine ,Bacterial Proteins ,Lactobacillus ,medicine ,lcsh:Science ,Multidisciplinary ,biology ,Bacteria ,Chemistry ,Vesicle ,Probiotics ,lcsh:R ,Membrane Proteins ,food and beverages ,biology.organism_classification ,Subcellular localization ,Lactobacillus acidophilus ,Lacticaseibacillus casei ,030104 developmental biology ,medicine.anatomical_structure ,Biochemistry ,lcsh:Q ,030217 neurology & neurosurgery ,Lactobacillus plantarum - Abstract
Bacterial membrane vesicles have been implicated in a broad range of functions in microbial communities from pathogenesis to gene transfer. Though first thought to be a phenomenon associated with Gram-negative bacteria, vesicle production in Staphylococcus aureus, Lactobacillus plantarum, and other Gram-positives has recently been described. Given that many Lactobacillus species are Generally Regarded as Safe and often employed as probiotics, the engineering of Lactobacillus membrane vesicles presents a new avenue for the development of therapeutics and vaccines. Here we characterize and compare the membrane vesicles (MVs) from three different Lactobacillus species (L. acidophilus ATCC 53544, L. casei ATCC 393, and L. reuteri ATCC 23272), with the aim of developing future strategies for vesicle engineering. We characterize the vesicles from each Lactobacillus species comparing the physiochemical properties and protein composition of each. More than 80 protein components from Lactobacillus-derived MVs were identified, including some that were enriched in the vesicles themselves suggesting vesicles as a vehicle for antimicrobial delivery. Additionally, for each species vesicular proteins were categorized based on biological pathway and examined for subcellular localization signals in an effort to identify possible sorting mechanisms for MV proteins.
- Published
- 2019
35. Cholesterol Functionalization of Gold Nanoparticles Enhances Photoactivation of Neural Activity
- Author
-
Francisco Bezanilla, João L. Carvalho-de-Souza, Igor Vurgaftman, David R. Pepperberg, Eunkeu Oh, James B. Delehanty, Alan L. Huston, and Okhil K. Nag
- Subjects
Physiology ,Cognitive Neuroscience ,Membrane lipids ,Metal Nanoparticles ,Biochemistry ,Article ,Rats, Sprague-Dawley ,Cell membrane ,03 medical and health sciences ,0302 clinical medicine ,Ganglia, Spinal ,medicine ,Animals ,Humans ,Ion channel ,030304 developmental biology ,Neurons ,0303 health sciences ,Chemistry ,Cell Membrane ,technology, industry, and agriculture ,Depolarization ,Cell Biology ,General Medicine ,Transmembrane protein ,Rats ,Cholesterol ,HEK293 Cells ,Membrane ,medicine.anatomical_structure ,Membrane protein ,Colloidal gold ,Biophysics ,Gold ,Photic Stimulation ,030217 neurology & neurosurgery - Abstract
Gold nanoparticles (AuNPs) attached to the extracellular leaflet of the plasma membrane of neurons can enable the generation of action potentials (APs) in response to brief pulses of light. Recently described techniques to stably bind AuNP bioconjugates directly to membrane proteins (ion channels) in neurons enable robust AP generation mediated by the photoexcited conjugate. However, a strategy that binds the AuNP to the plasma membrane in a non protein-specific manner could represent a simple, single-step means of establishing light-responsiveness in multiple types of excitable neurons contained in the same tissue. On the basis of the ability of cholesterol to insert into the plasma membrane, here we test whether AuNP functionalization with linear dihydrolipoic acid-poly(ethylene) glycol (DHLA-PEG) chains that are distally terminated with cholesterol (AuNP-PEG-Chol) can enable light-induced AP generation in neurons. Dorsal root ganglion (DRG) neurons of rat were labeled with 20 nm diameter spherical AuNP-PEG-Chol conjugates wherein ∼30% of the surface ligands (DHLA-PEG-COOH) were conjugated to PEG-Chol. Voltage recordings under current-clamp conditions showed that DRG neurons labeled in this manner exhibited a capacity for AP generation in response to microsecond and millisecond pulses of 532 nm light, a property attributable to the close tethering of AuNP-PEG-Chol conjugates to the plasma membrane facilitated by the cholesterol moiety. Light-induced AP and subthreshold depolarizing responses of the DRG neurons were similar to those previously described for AuNP conjugates targeted to channel proteins using large, multicomponent immunoconjugates. This likely reflected the AuNP-PEG-Chol's ability, upon plasmonic light absorption and resultant slight and rapid heating of the plasma membrane, to induce a concomitant transmembrane depolarizing capacitive current. Notably, AuNP-PEG-Chol delivered to DRG neurons by inclusion in the buffer contained in the recording pipet/electrode enabled similar light-responsiveness, consistent with the activity of AuNP-PEG-Chol bound to the inner (cytofacial) leaflet of the plasma membrane. Our results demonstrate the ability of AuNP-PEG-Chol conjugates to confer timely stable and direct responsiveness to light in neurons. Further, this strategy represents a general approach for establishing excitable cell photosensitivity that could be of substantial advantage for exploring a given tissue's suitability for AuNP-mediated photocontrol of neural activity.
- Published
- 2018
36. The SARS-CoV-2 Cytopathic Effect Is Blocked by Lysosome Alkalizing Small Molecules
- Author
-
Mark J. Henderson, Manisha Pradhan, Sam Michael, Miao Xu, Paul Shinn, Zina Itkin, Kirill Gorshkov, Mason A. Wolak, Min Shen, Carleen Klumpp-Thomas, Juan Carlos de la Torre, Robert Bostwick, Lynn Rasmussen, Catherine Z. Chen, Wei Zheng, Wenwei Huang, Yu-Shan Cheng, Matthew D. Hall, Khalida Shamim, Wei Zhu, Donald C. Lo, Kimihiro Susumu, Anton Simeonov, Bruce Nguyen Tran, Xin Hu, and Eunkeu Oh
- Subjects
0301 basic medicine ,autophagy ,Chemistry ,SARS-CoV-2 ,030106 microbiology ,Autophagy ,coronavirus ,Pharmacology ,medicine.disease_cause ,small molecule inhibitors ,Virus ,Article ,cytopathic effect ,03 medical and health sciences ,Drug repositioning ,030104 developmental biology ,medicine.anatomical_structure ,Infectious Diseases ,Chloroquine ,Lysosome ,medicine ,Vero cell ,Coronavirus ,medicine.drug ,Cytopathic effect - Abstract
Understanding the SARS-CoV-2 virus' pathways of infection, virus-host-protein interactions, and mechanisms of virus-induced cytopathic effects will greatly aid in the discovery and design of new therapeutics to treat COVID-19. Chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID-19, have multiple cellular effects including alkalizing lysosomes and blocking autophagy as well as exhibiting dose-limiting toxicities in patients. Therefore, we evaluated additional lysosomotropic compounds to identify an alternative lysosome-based drug repurposing opportunity. We found that six of these compounds blocked the cytopathic effect of SARS-CoV-2 in Vero E6 cells with half-maximal effective concentration (EC50) values ranging from 2.0 to 13 μM and selectivity indices (SIs; SI = CC50/EC50) ranging from 1.5- to >10-fold. The compounds (1) blocked lysosome functioning and autophagy, (2) prevented pseudotyped particle entry, (3) increased lysosomal pH, and (4) reduced (ROC-325) viral titers in the EpiAirway 3D tissue model. Consistent with these findings, the siRNA knockdown of ATP6V0D1 blocked the HCoV-NL63 cytopathic effect in LLC-MK2 cells. Moreover, an analysis of SARS-CoV-2 infected Vero E6 cell lysate revealed significant dysregulation of autophagy and lysosomal function, suggesting a contribution of the lysosome to the life cycle of SARS-CoV-2. Our findings suggest the lysosome as a potential host cell target to combat SARS-CoV-2 infections and inhibitors of lysosomal function could become an important component of drug combination therapies aimed at improving treatment and outcomes for COVID-19.
- Published
- 2020
37. Affinity Immobilization of Semiconductor Quantum Dots and Metal Nanoparticles on Cellulose Paper Substrates
- Author
-
W. Russ Algar, Michael V. Tran, Igor L. Medintz, Olga Solodova, Kimihiro Susumu, Eleonora Petryayeva, Eunkeu Oh, and Hyungki Kim
- Subjects
Materials science ,technology, industry, and agriculture ,Nanoparticle ,Dithiol ,Nanotechnology ,02 engineering and technology ,Surface-enhanced Raman spectroscopy ,equipment and supplies ,010402 general chemistry ,021001 nanoscience & nanotechnology ,Platinum nanoparticles ,7. Clean energy ,01 natural sciences ,0104 chemical sciences ,chemistry.chemical_compound ,Cellulose fiber ,chemistry ,Colloidal gold ,Microcontact printing ,General Materials Science ,Cellulose ,0210 nano-technology - Abstract
Colloidal semiconductor quantum dots (QDs), metal nanoparticles, and cellulose paper are materials with numerous applications in bioanalysis and beyond. The functional properties of QDs and metal NPs are substantially different than those of cellulose, such that their integration with cellulose paper is potentially enabling for many applications. Here, we characterize and evaluate multiple chemistries that modify cellulose paper substrates for the affinity-based immobilization of QDs, gold nanoparticles (Au NPs), and platinum nanoparticles (Pt NPs). These chemistries include grafting of cellulose fibers with imidazole and dithiol groups, as well as the aminosilanization of cellulose fibers (both with and without subsequent grafting with dithiol groups). Cellulose modifications and nanoparticle immobilization are characterized by multiple techniques, including, but not limited to, X-ray photoelectron spectroscopy, scanning electron microscopy, and optical imaging, extinction, and fluorescence measurements. We demonstrate the on-paper immobilization of color-tuned mixtures of QDs, on-paper patterning of QDs by microcontact printing, and post-immobilization enhancement of energy transfer and model assays of protease activity. The robustness of QD photoluminescence is also evaluated between immobilization chemistries. Paper-immobilized Au NPs and Pt NPs are evaluated as potential substrates for SERS and as supported catalysts for a model decolorization reaction. Our cumulative results indicate that there may not be a one-size-fits-all immobilization chemistry. Instead, the immobilization chemistry should be tailored and optimized for the downstream application.
- Published
- 2020
38. Quantum Dot-Conjugated SARS-CoV-2 Spike Pseudo-Virions Enable Tracking of Angiotensin Converting Enzyme 2 Binding and Endocytosis
- Author
-
Mason A. Wolak, Wei Zhu, Manisha Pradhan, Miao Xu, Eunkeu Oh, Xin Hu, Kimihiro Susumu, Jiji Chen, Joyce C. Breger, and Kirill Gorshkov
- Subjects
Cell ,Pneumonia, Viral ,General Physics and Astronomy ,Metal Nanoparticles ,02 engineering and technology ,Plasma protein binding ,Peptidyl-Dipeptidase A ,010402 general chemistry ,Endocytosis ,01 natural sciences ,Article ,law.invention ,angiotensin converting enzyme 2 ,Betacoronavirus ,law ,Quantum Dots ,medicine ,Humans ,endocytosis ,General Materials Science ,Receptor ,Pandemics ,Quenching (fluorescence) ,receptor binding domain ,Chemistry ,SARS-CoV-2 ,General Engineering ,Virion ,COVID-19 ,quantum dot ,Transfection ,spike ,021001 nanoscience & nanotechnology ,0104 chemical sciences ,medicine.anatomical_structure ,Colloidal gold ,Spike Glycoprotein, Coronavirus ,Biophysics ,Recombinant DNA ,Angiotensin-Converting Enzyme 2 ,Gold ,fluorescence ,0210 nano-technology ,Coronavirus Infections ,hormones, hormone substitutes, and hormone antagonists ,Protein Binding - Abstract
The first step of SARS-CoV-2 infection is binding of the spike protein's receptor binding domain to the host cell's ACE2 receptor on the plasma membrane. Here, we have generated a versatile imaging probe using recombinant Spike receptor binding domain conjugated to fluorescent quantum dots (QDs). This probe is capable of engaging in energy transfer quenching with ACE2-conjugated gold nanoparticles to enable monitoring of the binding event in solution. Neutralizing antibodies and recombinant human ACE2 blocked quenching, demonstrating a specific binding interaction. In cells transfected with ACE2-GFP, we observed immediate binding of the probe on the cell surface followed by endocytosis. Neutralizing antibodies and ACE2-Fc fully prevented binding and endocytosis with low nanomolar potency. Importantly, we will be able to use this QD nanoparticle probe to identify and validate inhibitors of the SARS-CoV-2 Spike and ACE2 receptor binding in human cells. This work enables facile, rapid, and high-throughput cell-based screening of inhibitors for coronavirus Spike-mediated cell recognition and entry.
- Published
- 2020
39. Utilizing meta-analysis to understand the cellular toxicity of cadmium containing quantum dots
- Author
-
Eunkeu Oh and Igor L. Medintz
- Published
- 2020
40. A Multiparametric Evaluation of Quantum Dot Size and Surface-Grafted Peptide Density on Cellular Uptake and Cytotoxicity
- Author
-
Bella B. Manshian, Christy Maksoudian, Kimihiro Susumu, Stefaan J. Soenen, Igor L. Medintz, and Eunkeu Oh
- Subjects
Chemistry, Multidisciplinary ,Pharmaceutical Science ,Nanoparticle ,02 engineering and technology ,Cell-Penetrating Peptides ,01 natural sciences ,Mice ,BIODISTRIBUTION ,Materials Testing ,Cytotoxicity ,INTRACELLULAR DELIVERY ,chemistry.chemical_classification ,DAMAGE ,Chemistry ,LOCALIZATION ,021001 nanoscience & nanotechnology ,Mitochondria ,Protein Transport ,Colloidal gold ,Physical Sciences ,Nanomedicine ,tat Gene Products, Human Immunodeficiency Virus ,0210 nano-technology ,Life Sciences & Biomedicine ,Biotechnology ,Biodistribution ,Biochemistry & Molecular Biology ,Biocompatibility ,Cell Survival ,Surface Properties ,Biomedical Engineering ,Chemistry, Organic ,Bioengineering ,Biochemical Research Methods ,NANOMEDICINE ,Cell Line ,CADMIUM ,Quantum Dots ,Autophagy ,Animals ,Particle Size ,Pharmacology ,Reactive oxygen species ,Science & Technology ,010405 organic chemistry ,Organic Chemistry ,IN-VITRO ,0104 chemical sciences ,Quantum dot ,GOLD NANOPARTICLES ,CELLS ,Biophysics ,PENETRATING PEPTIDES ,Reactive Oxygen Species - Abstract
Despite the progress in nanotechnology for biomedical applications, great efforts are still being employed in optimizing nanoparticle (NP) design parameters to improve functionality and minimize bionanotoxicity. In this study, we developed CdSe/CdS/ZnS core/shell/shell quantum dots (QDs) that are compact ligand-coated and surface-functionalized with an HIV-1-derived TAT cell-penetrating peptide (CPP) analog to improve both biocompatibility and cellular uptake. Multiparametric studies were performed in different mammalian and murine cell lines to compare the effects of varying QD size and number of surface CPPs on cellular uptake, viability, generation of reactive oxygen species, mitochondrial health, cell area, and autophagy. Our results showed that the number of cell-associated NPs and their respective toxicity are higher for the larger QDs. Meanwhile, increasing the number of surface CPPs also enhanced cellular uptake and induced cytotoxicity through the generation of mitoROS and autophagy. Thus, here we report the optimal size and surface CPP combinations for improved QD cellular uptake. ispartof: BIOCONJUGATE CHEMISTRY vol:31 issue:4 pages:1077-1087 ispartof: location:United States status: published
- Published
- 2020
41. Competitive binding of gold nanospheres and nanorods on DNA origami substrates
- Author
-
William P. Klein, Igor L. Medintz, Eunkeu Oh, Sebastián A. Díaz, Divita Mathur, Jawad Naciri, Jake Fontana, and Hieu Bui
- Subjects
Materials science ,Nanostructure ,Colloidal gold ,DNA nanotechnology ,Nanoparticle ,DNA origami ,Nanotechnology ,Nanorod ,Surface plasmon resonance ,Biosensor - Abstract
DNA-directed assembly of gold nanoparticles into precise two- and three-dimensional patterns has enabled bold advances in probing their optical properties such as the local enhancement in their surface plasmon resonance. DNA nanostructures synthesized using the principles of DNA origami have been programmed to contain unique capture sites for positioning metal nanoparticles in diverse geometries for applications in biosensing, therapy, and miniature electronics. However, to enable scalability beyond simple 2-3 nanoparticle architectures, it is important to understand the requirement for orthogonal capture sequences for attaching more than a single gold nanoparticle on a DNA nanostructure. In this work, we sought to assemble an angular gold nanorod-nanosphere-nanorod pattern on a DNA origami triangle with multiple capture sites utilizing a common capture sequence. Results indicate that gold nanospheres preferentially bound to all the capture sites on the DNA origami triangle and prevented attachment of gold nanorods. This suggests that requirement for orthogonal capture sites is correlated with the physical properties of the individual nanoparticle such as shape and size.
- Published
- 2020
42. Melanin Produced by the Fast-Growing Marine Bacterium Vibrio natriegens through Heterologous Biosynthesis: Characterization and Application
- Author
-
Evan R. Glaser, Amy Chen, Dagmar H. Leary, Weiyao Li, Zachary Schultzhaus, Tanya Tschirhart, Jaimee R. Compton, Gary J. Vora, Erin E. Kelly, Daniel A. Phillips, Ali Dhinojwala, Pamela F. Lloyd, Zheng Wang, Paul T. Charles, Gregory F. Payne, Eunkyoung Kim, Okhil K. Nag, and Eunkeu Oh
- Subjects
Tyrosinase ,Heterologous ,Vibrio natriegens ,Applied Microbiology and Biotechnology ,Melanin ,03 medical and health sciences ,chemistry.chemical_compound ,Synthetic biology ,Biopolymers ,030304 developmental biology ,Vibrio ,Melanins ,0303 health sciences ,Natural product ,Ecology ,biology ,integumentary system ,030306 microbiology ,Monophenol Monooxygenase ,DHICA ,biology.organism_classification ,chemistry ,Biochemistry ,Bacillus megaterium ,Microorganisms, Genetically-Modified ,Bacteria ,Food Science ,Biotechnology - Abstract
Melanin is a pigment produced by organisms throughout all domains of life. Due to its unique physicochemical properties, biocompatibility, and biostability, there has been an increasing interest in the use of melanin for broad applications. In the vast majority of studies, melanin has been either chemically synthesized or isolated from animals, which has restricted its use to small-scale applications. Using bacteria as biocatalysts is a promising and economical alternative for the large-scale production of biomaterials. In this study, we engineered the marine bacterium Vibrio natriegens, one of the fastest-growing organisms, to synthesize melanin by expressing a heterologous tyrosinase gene and demonstrated that melanin production was much faster than in previously reported heterologous systems. The melanin of V. natriegens was characterized as a polymer derived from dihydroxyindole-2-carboxylic acid (DHICA) and, similarly to synthetic melanin, exhibited several characteristic and useful features. Electron microscopy analysis demonstrated that melanin produced from V. natriegens formed nanoparticles that were assembled as “melanin ghost” structures, and the photoprotective properties of these particles were validated by their protection of cells from UV irradiation. Using a novel electrochemical reverse engineering method, we observed that melanization conferred redox activity to V. natriegens. Moreover, melanized bacteria were able to quickly adsorb the organic compound trinitrotoluene (TNT). Overall, the genetic tractability, rapid division time, and ease of culture provide a set of attractive properties that compare favorably to current E. coli production strains and warrant the further development of this chassis as a microbial factory for natural product biosynthesis. IMPORTANCE Melanins are macromolecules that are ubiquitous in nature and impart a large variety of biological functions, including structure, coloration, radiation resistance, free radical scavenging, and thermoregulation. Currently, in the majority of investigations, melanins are either chemically synthesized or extracted from animals, which presents significant challenges for large-scale production. Bacteria have been used as biocatalysts to synthesize a variety of biomaterials due to their fast growth and amenability to genetic engineering using synthetic biology tools. In this study, we engineered the extremely fast-growing bacterium V. natriegens to synthesize melanin nanoparticles by expressing a heterologous tyrosinase gene with inducible promoters. Characterization of the melanin produced from V. natriegens-produced tyrosinase revealed that it exhibited physical and chemical properties similar to those of natural and chemically synthesized melanins, including nanoparticle structure, protection against UV damage, and adsorption of toxic compounds. We anticipate that producing and controlling melanin structures at the nanoscale in this bacterial system with synthetic biology tools will enable the design and rapid production of novel biomaterials for multiple applications.
- Published
- 2019
43. DNA–Nanoparticle Composites Synergistically Enhance Organophosphate Hydrolase Enzymatic Activity
- Author
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Scott A. Walper, Eunkeu Oh, Joyce C. Breger, Nabil Bassim, Anirban Samanta, Igor L. Medintz, and Kimihiro Susumu
- Subjects
0301 basic medicine ,chemistry.chemical_classification ,biology ,Nanoparticle ,Bioinorganic chemistry ,02 engineering and technology ,021001 nanoscience & nanotechnology ,Enzyme assay ,Turn (biochemistry) ,03 medical and health sciences ,chemistry.chemical_compound ,Synthetic biology ,030104 developmental biology ,Enzyme ,chemistry ,biology.protein ,General Materials Science ,A-DNA ,Composite material ,0210 nano-technology ,DNA - Abstract
Cell-free synthetic biology relies on optimally exploiting enzymatic activity, and recent demonstrations that nanoparticle (NP) and DNA scaffolding can enhance enzyme activity suggest new avenues toward this. A modular architecture consisting of a DNA cage displaying semiconductor quantum dots (QDs) that, in turn, ratiometrically display the organophosphate hydrolase phosphotriesterase (PTE) was utilized as a model system. Increasing DNA cage concentration relative to QD-PTE and creating a dense composite enhanced PTE rates up to 12.5-fold, suggesting strong synergy between the NP and DNA components; this putatively arises from increased enzymatic stability and alleviation of its rate-limiting step. Such bioinorganic composites may offer new scaffolding approaches for synthetic biology.
- Published
- 2018
44. Hybrid Liquid Crystal Nanocarriers for Enhanced Zinc Phthalocyanine-Mediated Photodynamic Therapy
- Author
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Okhil K. Nag, Jeffrey S. Erickson, Eunkeu Oh, Jawad Naciri, and James B. Delehanty
- Subjects
Indoles ,medicine.medical_treatment ,Biomedical Engineering ,Pharmaceutical Science ,chemistry.chemical_element ,Bioengineering ,Photodynamic therapy ,Nanotechnology ,02 engineering and technology ,Zinc ,Isoindoles ,010402 general chemistry ,01 natural sciences ,Drug Delivery Systems ,Liquid crystal ,Fluorescence Resonance Energy Transfer ,Organometallic Compounds ,medicine ,Humans ,Photosensitizer ,Pharmacology ,Zinc phthalocyanine ,Photosensitizing Agents ,Cell Membrane ,Organic Chemistry ,021001 nanoscience & nanotechnology ,Liquid Crystals ,0104 chemical sciences ,HEK293 Cells ,Photochemotherapy ,chemistry ,Zinc Compounds ,Nanoparticles ,Spectrophotometry, Ultraviolet ,Nanocarriers ,Reactive Oxygen Species ,0210 nano-technology ,Hydrophobic and Hydrophilic Interactions ,HeLa Cells ,Biotechnology - Abstract
Current challenges in photodynamic therapy (PDT) include both the targeted delivery of the photosensitizer (PS) to the desired cellular location and the maintenance of PS efficacy. Zinc phthalocyanine (ZnPc), a macrocyclic porphyrin and a potent PS for PDT, undergoes photoexcitation to generate reactive singlet oxygen that kills cells efficiently, particularly when delivered to the plasma membrane. Like other commonly employed PS, ZnPc is highly hydrophobic and prone to self-aggregation in aqueous biological media. Further, it lacks innate subcellular targeting specificity. Cumulatively, these attributes pose significant challenges for delivery via traditional systemic drug delivery modalities. Here, we report the development and characterization of a liquid crystal nanoparticle (LCNP)-based formulation for the encapsulation and targeted tethering of ZnPc to the plasma membrane bilayer. ZnPc was coloaded with the organic fluorophore, perylene (PY), in the hydrophobic polymeric matrix of the LCNP core. PY facilitated the fluorescence-based tracking of the LCNP carrier while also serving as a Förster resonance energy transfer (FRET) donor to the ZnPc acceptor. This configuration availed efficient singlet oxygen generation via enhanced excitation of ZnPc from multiple surrounding PY energy donors. When excited in a FRET configuration, cuvette-based assays revealed that singlet oxygen generation from the ZnPc was ∼1.8-fold greater and kinetically 12 times faster compared to when the ZnPc was excited directly. The specific tethering of the LCNPs to the plasma membrane of HEK 293 T/17 and HeLa cells was achieved by surface functionalization of the NPs with PEGylated cholesterol. In HeLa cells, LCNPs coloaded with PY and ZnPc, when photoexcited in a FRET configuration, mediated 70% greater cell killing compared to LCNPs containing ZnPc alone (direct excitation of ZnPc). This was attributed to a significant increase of the oxidative stress in the cells during the PDT. Overall, this work details the ability of the LCNP platform to facilitate (1) the specific tethering of the PY-ZnPc FRET pair to the plasma membrane and (2) the FRET-mediated, augmented singlet oxygen generation for enhanced PDT relative to the direct excitation of ZnPc alone.
- Published
- 2018
45. A Quantum Dot-Protein Bioconjugate That Provides for Extracellular Control of Intracellular Drug Release
- Author
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Scott A. Walper, Lauren D. Field, Guillermo Lasarte-Aragonés, Igor L. Medintz, Eunkeu Oh, James B. Delehanty, and Kimihiro Susumu
- Subjects
Biomedical Engineering ,Pharmaceutical Science ,Bioengineering ,02 engineering and technology ,010402 general chemistry ,Binding, Competitive ,01 natural sciences ,Maltose-Binding Proteins ,Maltose-binding protein ,Drug Delivery Systems ,Chlorocebus aethiops ,Quantum Dots ,Extracellular ,Animals ,Humans ,Maltose ,Pharmacology ,Binding Sites ,Bioconjugation ,biology ,Chemistry ,beta-Cyclodextrins ,Organic Chemistry ,021001 nanoscience & nanotechnology ,Ligand (biochemistry) ,0104 chemical sciences ,Drug Liberation ,Förster resonance energy transfer ,COS Cells ,Drug delivery ,Biophysics ,biology.protein ,0210 nano-technology ,Intracellular ,Biotechnology ,Conjugate - Abstract
The ability to control the intracellular release of drug cargos from nanobioconjugate delivery scaffolds is critical for the successful implementation of nanoparticle (NP)-mediated drug delivery. This is particularly true for hard NP carriers such as semiconductor quantum dots (QDs) and gold NPs. Here, we report the development of a QD-based multicomponent drug release system that, when delivered to the cytosol of mammalian cells, is triggered to release its drug cargo by the simple addition of a competitive ligand to the extracellular medium. The ensemble construct consists of the central QD scaffold that is decorated with a fixed number of maltose binding proteins (MBPs). The MBP binding site is loaded with dye or drug conjugates of the maltose analogue beta-cyclodextrin (βCD) to yield a QD-MBP-βCD ensemble conjugate. The fidelity of conjugate assembly is monitored by Förster resonance energy transfer (FRET) from the QD donor to the dye/drug acceptor. Microplate-based FRET assays demonstrated that the βCD conjugate was released from the MBP binding pocket by maltose addition with an affinity that matched native MBP-maltose binding interactions. In COS-1 cells, the microinjected assembled conjugates remained stably intact in the cytosol until the addition of maltose to the extracellular medium, which underwent facilitated uptake into the cell. Live cell FRET-based confocal microscopy imaging captured the kinetics of realtime release of the βCD ligand as a function of extracellular maltose concentration. Our results demonstrate the utility of the self-assembled QD-MBP-βCD system to facilitate intracellular drug release that is triggered extracellularly through the simple addition of a well-tolerated nutrient and is not dependent on the use of light, magnetic field, ultrasound, or other traditional methods of stimulated drug release. We expect this extracellularly triggered drug release modality to be useful for the in vitro characterization of new drug candidates intended for systemic delivery/actuation and potentially for on-demand drug release in vivo.
- Published
- 2018
46. Exploring attachment chemistry with FRET in hybrid quantum dot dye-labeled DNA dendrimer composites
- Author
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Susan Buckhout-White, Anirban Samanta, Igor L. Medintz, Eunkeu Oh, and Kimihiro Susumu
- Subjects
Biomedical Engineering ,Energy Engineering and Power Technology ,02 engineering and technology ,010402 general chemistry ,01 natural sciences ,Industrial and Manufacturing Engineering ,Dendrimer ,Materials Chemistry ,Chemical Engineering (miscellaneous) ,Composite material ,chemistry.chemical_classification ,business.industry ,Process Chemistry and Technology ,Biomolecule ,021001 nanoscience & nanotechnology ,0104 chemical sciences ,Förster resonance energy transfer ,chemistry ,Chemistry (miscellaneous) ,Covalent bond ,Quantum dot ,Photonics ,0210 nano-technology ,business ,Luminescence ,Biosensor - Abstract
Luminescent semiconductor quantum dots (QDs) and a range of biomolecules are now being routinely co-integrated into functional optical devices in pursuit of creating novel ‘value added’ photonic and energy harvesting/transfer materials. Amongst the biological molecules, structural DNA architectures are particularly useful due to their unrivaled ability to assume almost any desired shape along with allowing fluorophores to be precisely arranged on them with controlled stoichiometry and sub-nanometer positional accuracy. The unique properties available to joint QD–DNA composites suggest them for a host of new applications in light harvesting, biosensing, and molecular computation amongst others. To fully realize the synergistic benefits from such organic–inorganic composites, especially when they constitute complex, multidimensional Forster resonance energy transfer (FRET) networks, a detailed understanding of the mechanisms that govern the individual components is imperative. Here, we demonstrate hybrid FRET systems comprising an initial QD scaffold/donor displaying DNA dendrimers decorated with dyes and which are capable of efficiently capturing UV light and transporting it to spectrally and spatially distant fluorophores via multistep FRET. We evaluate two primary strategies to conjugate the DNA-dendrimers to the QDs, namely covalent attachment of DNA to the termini of the QDs surface ligands and polyhistidine-based metal affinity coordination of modified DNA to the QD's ZnS shell surface. Analysis of the resulting FRET data shows that the dendritic arrangement of the dyes and the ability to place multiple dendrimer copies around the QD's nontrivial surface provides for significant energy transfer efficiencies of 20–25% through these multi-FRET step systems. In analyzing the properties of the conjugates, we further find that each assembly chemistry brings with it a series of benefits and liabilities that serve as mutual trade-offs and potential rules of thumb for designing future nanodevices based on these materials.
- Published
- 2018
47. Utility of PEGylated dithiolane ligands for direct synthesis of water-soluble Au, Ag, Pt, Pd, Cu and AuPt nanoparticles
- Author
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W. Russ Algar, Eunkeu Oh, Ramasis Goswami, James B. Delehanty, Igor L. Medintz, Kimihiro Susumu, and Christopher A. Klug
- Subjects
Denticity ,Ligand ,technology, industry, and agriculture ,Metals and Alloys ,Nanoparticle ,02 engineering and technology ,General Chemistry ,010402 general chemistry ,021001 nanoscience & nanotechnology ,01 natural sciences ,Combinatorial chemistry ,Catalysis ,0104 chemical sciences ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Dithiolane ,chemistry.chemical_compound ,Water soluble ,chemistry ,PEG ratio ,Drug delivery ,Materials Chemistry ,Ceramics and Composites ,0210 nano-technology - Abstract
Water soluble metallic nanoparticles are being developed for a variety of roles ranging from catalysis to drug delivery and as potential contrast agents. We demonstrate direct synthesis of high-quality water-soluble Au, Ag, Pt, Pd, Cu and alloyed AuPt nanoparticles as well as ligand-exchange of FePt, cubic Pt and Au/Pt core/shell nanoparticles using bidentate dithiolane PEG as a universal ligand. Simple chemistry can greatly expand the applications of metal nanoparticles.
- Published
- 2018
48. In Situ Self‐Assembly of Quantum Dots at the Plasma Membrane Mediates Energy Transfer‐Based Activation of Channelrhodopsin (Part. Part. Syst. Charact. 7/2021)
- Author
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Michael H. Stewart, Okhil K. Nag, Kimihiro Susumu, Joseph R. Spangler, Megan E. Muroski, Eunkeu Oh, Lauren D. Field, James B. Delehanty, and Scott A. Walper
- Subjects
Membrane potential ,Materials science ,biology ,Channelrhodopsin ,General Chemistry ,Condensed Matter Physics ,Fluorescence ,Förster resonance energy transfer ,Membrane ,Quantum dot ,Rhodopsin ,biology.protein ,Biophysics ,General Materials Science ,Self-assembly - Published
- 2021
49. In Situ Self‐Assembly of Quantum Dots at the Plasma Membrane Mediates Energy Transfer‐Based Activation of Channelrhodopsin
- Author
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James B. Delehanty, Joseph R. Spangler, Scott A. Walper, Okhil K. Nag, Kimihiro Susumu, Megan E. Muroski, Michael H. Stewart, Eunkeu Oh, and Lauren D. Field
- Subjects
Membrane potential ,biology ,Chemistry ,Channelrhodopsin ,General Chemistry ,Condensed Matter Physics ,Fluorescence ,Membrane ,Förster resonance energy transfer ,Quantum dot ,Rhodopsin ,Biophysics ,biology.protein ,General Materials Science ,Self-assembly - Published
- 2021
50. Purple-, Blue-, and Green-Emitting Multishell Alloyed Quantum Dots: Synthesis, Characterization, and Application for Ratiometric Extracellular pH Sensing
- Author
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Kimihiro Susumu, Lauren D. Field, Igor L. Medintz, James B. Delehanty, Valle Palomo, Philip E. Dawson, Alan L. Huston, Eunkeu Oh, and Michael Hunt
- Subjects
Aqueous solution ,Ligand ,Chemistry ,General Chemical Engineering ,Analytical chemistry ,02 engineering and technology ,General Chemistry ,010402 general chemistry ,021001 nanoscience & nanotechnology ,Photochemistry ,01 natural sciences ,Fluorescence ,0104 chemical sciences ,Ion ,Full width at half maximum ,Quantum dot ,Materials Chemistry ,Surface modification ,0210 nano-technology ,Luminescence - Abstract
We report the synthesis of a series of CdxZn1–xSe/CdyZn1–yS/ZnS and ZnSe/CdyZn1–yS/ZnS multishell alloyed luminescent semiconductor quantum dots (QDs) with fluorescence maxima ranging from 410 to 530 nm which cover the purple, blue, and green portion of the spectrum. Their subsequent surface modification to prepare water-soluble blue-emitting QDs, characterization, and application for ratiometric pH sensing in aqueous buffers and in an extracellular environment are further described. QDs were synthesized starting from ZnSe cores, and the fluorescence peak positions were tuned by (i) cation exchange with cadmium ions and/or (ii) overcoating with CdyZn1–yS layers. The as-prepared QDs had reasonably high fluorescence quantum yields (∼30–55%), narrow fluorescence bands (fwhm ∼25–35 nm), and monodispersed semispherical shapes. Ligand exchange with hydrophilic compact ligands was successfully carried out to prepare a series of water-soluble blue-emitting QDs. QDs coated with the hydrophilic compact ligands pres...
- Published
- 2017
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