Dr. Guillermo Lasarte-Aragonés Profile

Dr. Guillermo Lasarte-Aragonés

at US Naval Research Lab

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Publications (2)

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Proceedings Article | 7 March 2019 Presentation + Paper

Time-gated FRET from terbium labeled antibodies to quantum dot acceptors for broad ADP sensing

Sebastián Díaz, Guillermo Lasarte-Aragonés, Robert Lowery, James Vranish, William Klein, Kimihiro Susumu, Igor Medintz

Proceedings Volume 10892, 108920P (2019) https://doi.org/10.1117/12.2508626

KEYWORDS: Fluorescence resonance energy transfer, Terbium, Luminescence, Sensors, Quantum dots, Signal detection

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Time-gated Förster resonance energy transfer (TR-FRET) introduces a time-gate before the detection of the fluorescence spectra or photon count. If the donor is sufficiently long-lived TR-FRET allows for any initial acceptor sensitization to decay before the measurement. TR-FRET in the μs range is particularly advantageous for small molecule assays as it eliminates background fluorescence from screening compounds, which typically have ns lifetimes. The sensor we developed utilizes Terbium (Tb)-labeled antibodies (Ab) that selectively recognizes adenosine diphosphate (ADP). The Tb emitters have fluorescence lifetimes on the ms scale, making them excellent candidates for TR-FRET donors. In an attempt to increase the FRET signal we utilized a semiconductor quantum dot (QD) as an acceptor. The QD presented an ADP modified His6-peptide conjugated to its surface via self-assembly metal-affinity coordination, which bound the Tb labeled Ab to the QD surface. QDs have large extinction coefficients, broad absorption, brightness, and sharp emission peaks, optimal for sensitive and multiplexed detection. By using a QD acceptor the Förster radius was increased by approximately 2 nm as compared to traditional organic dyes. We were able to demonstrate a Tb-to-QD based TR-FRET bioassay for broadly applicable ADP sensing, working at nM concentrations for sensor, analyte, and enzyme. Quantitative values were obtained for the kinetics of a model enzyme (glucokinase). The specific sensor was also capable of discriminating enzyme inhibitor capabilities of structurally similar compounds. The strategy of using modified peptides to present antibody epitopes on QD surfaces is readily transferable to other assays.

Proceedings Article | 23 February 2018 Presentation + Paper

Nanoparticle bioconjugate for controlled cellular delivery of doxorubicin

Ajmeeta Sangtani, Eleonora Petryayeva, Miao Wu, Kimihiro Susumu, Eunkeu Oh, Alan Huston, Guillermo Lasarte-Aragones, Igor Medintz, W. Russ Algar, James Delehanty

Proceedings Volume 10507, 105070M (2018) https://doi.org/10.1117/12.2290031

KEYWORDS: Toxicity, Nanoparticles, Modulation, Chemistry, Control systems, Quantum dots

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Nanoparticle (NP)-mediated drug delivery offers the potential to overcome limitations of systemic delivery, including the ability to specifically target cargo and control release of NP-associated drug cargo. Doxorubicin (DOX) is a widely used FDA-approved cancer therapeutic; however, multiple side effects limit its utility. Thus, there is wide interest in modulating toxicity after cell delivery. Our goal here was to realize a NP-based DOX-delivery system that can modulate drug toxicity by controlling the release kinetics of DOX from the surface of a hard NP carrier. To achieve this, we employed a quantum dot (QD) as a central scaffold which DOX was appended via three different peptidyl linkages (ester, disulfide, hydrazone) that are cleavable in response to various intracellular conditions. Attachment of a cell penetrating peptide (CPP) containing a positively charged polyarginine sequence facilitates endocytosis of the ensemble. Polyhistidine-driven metal affinity coordination was used to self-assemble both peptides to the QD surface, allowing for fine control over both the ratio of peptides attached to the QD as well as DOX dose delivered to cells. Microplate-based Förster resonance energy transfer assays confirmed the successful ratiometric assembly of the conjugates and functionality of the linkages. Cell delivery experiments and cytotoxicity assays were performed to compare the various cleavable linkages to a control peptide where DOX is attached through an amide bond. The role played by various attachment chemistries used in QD-peptide-drug assemblies and their implications for the rationale in design of NPbased constructs for drug delivery is described here.

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