RESUMEN
Two fluorescent dyes covalently attached in diagonal interstrand orientation to siRNA undergo energy transfer and thereby enable a dual color fluorescence readout (red/green) for hybridization. Three different structural variations were carried out and compared by their optical properties, including (i) the base surrogate approach with an acyclic linker as a substitute of the 2-deoxyriboside between the phosphodiester bridges, (ii) the 2'-modification of conventional ribofuranosides and (iii) the arabino-configured 2'-modification. The double stranded siRNA with the latter type of modification delivered the best energy transfer efficiency, which was explained by molecular dynamics simulations that showed that the two dyes are more flexible at the arabino-configured sugars compared to the completely stacked situation at the ribo-configured ones. Single molecule fluorescence lifetime measurements indicate their application in fluorescence cell imaging, which reveals a red/green fluorescence contrast in particular for the arabino-configured 2'-modification by the two dyes, which is key for tracking of siRNA transport into HeLa cells.
Asunto(s)
Colorantes Fluorescentes/química , Microscopía Confocal , ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismo , Secuencia de Bases , Transporte Biológico , Color , Células HeLa , Humanos , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , ARN Interferente Pequeño/genéticaRESUMEN
A competitive binding assay based on localized surface plasmon resonance (LSPR) of folic acid-functionalized gold nanoparticles (FA-AuNPs) and human dihydrofolate reductase enzyme (hDHFR) was developed to detect nanomolar to micromolar concentrations of the widely applied anti-cancer drug, methotrexate (MTX). By the nature of the competitive assay for MTX, the LSPR shift from specific binding between FA-AuNPs and the free enzyme was inversely proportional to the concentration of MTX. In addition, the dynamic range for MTX was tuned from 10(-11) to 10(-6) M by varying the concentration of hDHFR from 1 to 100 nM. Inter-day reproducibility and recovery of MTX spiked in phosphate buffer saline (PBS) were excellent. Potential interferents such as FA, trimethoprim (TMP) and 4-amino-4-deoxy-N-methylpteroic acid (DAMPA) did not occur in the concentration range of interest for MTX. Clinical samples of human serum from patients undergoing MTX chemotherapy were analyzed following a simple solid-phase extraction step to isolate MTX from the serum matrix, with a limit of detection of 155 nM. Validation of the LSPR method was carried out in comparison to Fluorescence Polarization Immunoassay (FPIA), a commonly used method in clinical settings, and LC-MS/MS, a reference technique. The results of the LSPR competitive assay compared well to FPIA and LC-MS/MS, with a slope of 2.4 and 1.1, respectively, for the correlation plots. The method established herein is intended for therapeutic drug monitoring (TDM) of MTX levels in patients undergoing chemotherapy to ensure safety and efficacy of the treatment.
Asunto(s)
Antimetabolitos Antineoplásicos/sangre , Metotrexato/sangre , Resonancia por Plasmón de Superficie , Antimetabolitos Antineoplásicos/aislamiento & purificación , Antimetabolitos Antineoplásicos/uso terapéutico , Cromatografía Líquida de Alta Presión , Monitoreo de Drogas , Inmunoensayo de Polarización Fluorescente , Ácido Fólico/química , Oro/química , Humanos , Nanopartículas del Metal/química , Metotrexato/aislamiento & purificación , Metotrexato/uso terapéutico , Neoplasias/tratamiento farmacológico , Extracción en Fase Sólida , Espectrometría de Masas en Tándem , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
We have studied the effect of the zwitterionic surface coating of quantum dots (QDs) on their interaction with a serum supplemented cell medium and their internalization by human cervical carcinoma (HeLa) cells. Zwitterionic QDs showed negligible adsorption of human serum albumin (HSA) selected as a model serum protein, in contrast to similar but negatively charged QDs. The incorporation of zwitterionic QDs by HeLa cells was found to be lower than for negatively charged QDs and for positively charged QDs, for which the uptake yield was largest. Our results suggest that the suppression of protein adsorption, here accomplished by zwitterionic QD surfaces, offers a strategy that allows for reducing the cellular uptake of nanoparticles.