RESUMO
Upconversion nanoparticles (UCNPs) should be particularly well suited for measurement inside cells because they can be imaged down to submicrometer dimensions in near real time using fluorescence microscopy, and they overcome problems, such as photobleaching, autofluorescence, and deep tissue penetration, that are commonly encountered in cellular imaging applications. In this study, the performance of an UCNP modified with a pH-sensitive dye (pHAb) is studied. The dye (emission wavelength 580 nm) was attached in a polyethylene imine (PEI) coating on the UCNP and excited via the 540-nm UCNP emission under 980-nm excitation. The UC resonance energy transfer efficiencies at different pHs ranged from 25 to 30% and a Förster distance of 2.56 nm was predicted from these results. Human neuroblastoma SH-SY5Y cells, equilibrated with nigericin H+/K+ ionophore to equalize the intra- and extracellular pH' showed uptake of the UCNP-pHAb conjugate particles and, taking the ratio of the intensity collected from the pHAb emission channel (565-630 nm) to that from the UCNP red emission channel (640-680 nm), produced a sigmoidal pH response curve with an apparent pKa for the UCNP-pHAb of ~ 5.1. The UCNP-pHAb were shown to colocalize with LysoBrite dye, a lysosome marker. Drug inhibitors such as chlorpromazine (CPZ) and nystatin (NYS) that interfere with clathrin-mediated endocytosis and caveolae-mediated endocytosis, respectively, were investigated to elucidate the mechanism of nanoparticle uptake into the cell. This preliminary study suggests that pH indicator-modified UCNPs such as UCNP-pHAb can report pH in SH-SY5Y cells and that the incorporation of the nanoparticles into the cell occurs via clathrin-mediated endocytosis. Graphical abstract.
Assuntos
Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Nanopartículas/química , Linhagem Celular , Humanos , Concentração de Íons de Hidrogênio , Microscopia de Fluorescência/métodos , Nanopartículas/ultraestruturaRESUMO
Upconversion nanoparticles (UCNPs) can provide a vehicle for chemical imaging by coupling chemically sensitive dyes and quenchers. The mechanism for coupling of two anthraquinone dyes, Calcium Red and Alizarin Red S, was investigated as a function of pH. The green emission band of the UCNPs was quenched by a pH-dependent inner filter effect (IFE) while the red emission band remained unchanged and acted as the reference signal for ratiometric pH measurements. Contrary to previous expectation, there was little evidence for a resonance energy transfer (RET) mechanism even when the anthraquinones were attached onto the UCNPs through electrostatic attraction. Since the UCNPs are point emitters, only emitters close to the surface of the UCNP are within the expected Förster distance and UC-RET is <10%. The theoretical and experimental analysis of the interaction between UCNPs and pH-sensitive quenchers will allow the design of UCNP pH sensors for determination of pH via IFE.
RESUMO
BACKGROUND: Cancer is first and foremost a disease of the genome. Specific genetic signatures within a tumour are prognostic of disease outcome, reflect subclonal architecture and intratumour heterogeneity, inform treatment choices and predict the emergence of resistance to targeted therapies. Minimally invasive liquid biopsies can give temporal resolution to a tumour's genetic profile and allow the monitoring of treatment response through levels of circulating tumour DNA (ctDNA). However, the detection of ctDNA in repeated liquid biopsies is currently limited by economic and time constraints associated with targeted sequencing. METHODS: Here we bioinformatically profile the mutational and copy number spectrum of The Cancer Genome Network's lung adenocarcinoma dataset to uncover recurrently mutated genomic loci. RESULTS: We build a panel of 400 hotspot mutations and show that the coverage extends to more than 80% of the dataset at a median depth of 8 mutations per patient. Additionally, we uncover several novel single-nucleotide variants present in more than 5% of patients, often in genes not commonly associated with lung adenocarcinoma. CONCLUSION: With further optimisation, this hotspot panel could allow molecular diagnostics laboratories to build curated primer banks for 'off-the-shelf' monitoring of ctDNA by droplet-based digital PCR or similar techniques, in a time- and cost-effective manner.