Photosensitiser functionalised luminescent upconverting nanoparticles for efficient photodynamic therapy of breast cancer cells.

Photodynamic therapy (PDT) is a well-established treatment of cancer in which cell toxic reactive oxygen species, including singlet oxygen (1O2), are produced by a photosensitiser drug following irradiation of a specific wavelength. Visible light is commonly used as the excitation source in PDT, although these wavelengths do have limited tissue penetration. In this research, upconverting nanoparticles (UCNPs) functionalised with the photosensitiser Rose Bengal (RB) have been designed and synthesised for PDT of breast cancer cells. The use of UCNPs shifts the required excitation wavelength for the production of 1O2 to near infrared light (NIR) thus allowing deeper tissue penetration. The system was designed to maximise the production of 1O2via efficient Förster resonance energy transfer (FRET) from the UCNPs to the photosensitiser. Highly luminescent NaYF4:Yb,Er,Gd@NaYF4 core-shell UCNPs were synthesised that exhibited two main anti-Stokes emission bands at 541 and 652 nm following 980 nm irradiation. RB was chosen as the photosensitiser since its absorption band overlaps with the green emission of the UCNPs. To achieve efficient energy transfer from the nanoparticles to the photosensitiser, the functionalised UCNPs included a short l-lysine linker to attach the RB to the nanocore yielding RB-lysine functionalised UCNPs. The efficient FRET from the UCNPs to the RB was confirmed by luminescence lifetime measurements. The light emitted by the UCNPs at 541 nm, following excitation at 980 nm, generates the 1O2via the RB. Multi-photon and confocal laser scanning microscopies confirmed the internalisation of the RB-lysine-UCNPs by SK-BR-3 breast cancer cells. Cell viability studies revealed that the RB-lysine-UCNPs induced low dark toxicity in cells prior to PDT treatment. Importantly, following irradiation at 980 nm, high levels of cell death were observed in cells loaded with the RB-lysine-UCNPs. Cell death following PDT treatment was also confirmed using propidium iodide and confocal microscopy. The high drug loading capacity (160 RB/nanoparticle) of the UCNPs, the efficient FRET from the UCNPs to the photosensitiser, the high level of accumulation inside the cells and their PDT cell kill suggest that the RB-lysine-UCNPs are promising for NIR PDT and hence suitable for the treatment of deep-lying cancer tumours.

Particle-Size-Dependent Förster Resonance Energy Transfer from Upconversion Nanoparticles to Organic Dyes.

Upconversion nanoparticles (UCNPs) are attractive candidates for energy transfer-based analytical applications. In contrast to classical donor-acceptor pairs, these particles contain many emitting lanthanide ions together with numerous acceptor dye molecules at different distances to each other, strongly depending on the particle diameter. UCNPs with precisely controlled sizes between 10 and 43 nm were prepared and functionalized with rose bengal and sulforhodamine B by a ligand-exchange procedure. Time-resolved studies of the upconversion luminescence of the UCNP donor revealed a considerable shortening of the donor lifetime as a clear hint for Förster resonance energy transfer (FRET). FRET was most pronounced for 21 nm-sized UCNPs, yielding a FRET efficiency of 60%. At larger surface-to-volume ratios, the FRET efficiency decreased by an increasing competition of nonradiative surface deactivation. Such dye-UCNP architectures can also provide an elegant way to shift the UCNP emission color, since the fluorescence intensity of the organic dyes excited by FRET was comparable to that of the upconversion emission of smaller particles.

Targeted Single Particle Tracking with Upconverting Nanoparticles.

Single particle tracking (SPT) is a powerful technique for real-time microscopic visualization of the movement of individual biomolecules within or on the surface of living cells. However, SPT often suffers from the suboptimal performance of the photon-emitting labels used to tag the biomolecules of interest. For example, fluorescent dyes have poor photostability, while quantum dots suffer from blinking that hampers track acquisition and interpretation. Upconverting nanoparticles (UCNPs) have recently emerged as a promising anti-Stokes luminescent label for SPT. In this work, we demonstrated targeted SPT using UCNPs. For this, we synthesized 30 nm diameter doped UCNPs and coated them with amphiphilic polymers decorated with polyethylene glycol chains to make them water-dispersible and minimize their nonspecific interactions with cells. Coated UCNPs highly homogeneous in brightness (as confirmed by a single particle investigation) were functionalized by immunoglobulin E (IgE) using a biotin-streptavidin strategy. Using these IgE-UCNP SPT labels, we tracked high-affinity IgE receptors (FcεRI) on the membrane of living RBL-2H3 mast cells at 37 °C in the presence and absence of antigen and obtained good agreement with the literature. Moreover, we used the FcεRI-IgE receptor-antibody system to directly compare the performance of UCNP-based SPT labels to organic dyes (AlexaFluor647) and quantum dots (QD655). Due to their photostability as well as their backgroundless and continuous luminescence, SPT trajectories obtained with UCNP labels are no longer limited by the photophysics of the label but only by the dynamics of the system and, in particular, the movement of the label out of the field of view and/or focal plane.

Distinctive FDG-PET/CT Findings in Acute Neurological Hospital Care.

A compilation of 6 distinctive 18F-fluorodeoxyglucose positron emission tomography (PET) combined with computed tomography (CT) findings in the acute setting of neurohospital care is presented. In case 1, PET/CT allowed the final diagnosis of circumscribed ischemic infarction by demonstrating a clear pattern of luxury perfusion. In case 2, diagnosis of thalamic abscess was made, whereby PET/CT demonstrated an empty zone. Hypermetabolic enlarged hilar lymph nodes and hypermetabolic spinal lumbar roots in PET/CT led to the diagnosis of neurosarcoidosis in case 3. In case 4, a hypermetabolic brain focus in PET/CT identified the seizure focus in epilepsia partialis continua. A cerebral hemispheric hypometabolism in PET/CT in case 5 supported the diagnosis of Creutzfeldt-Jakob disease, which initially mimicked acute stroke. In case 6, PET/CT detected infective endocarditis as a source of multiple cerebral ischemic lesions. In conclusion, PET/CT can contribute importantly to find the correct diagnosis in acute neurohospital patients.

Time-dependent luminescence loss for individual upconversion nanoparticles upon dilution in aqueous solution.

Single-particle luminescence microscopy is a powerful method to extract information on biological systems that is not accessible by ensemble-level methods. Upconversion nanoparticles (UCNPs) are a particularly promising luminophore for single-particle microscopy as they provide stable, non-blinking luminescence and allow the avoidance of biological autofluorescence by their anti-Stokes emission. Recently, ensemble measurements of diluted aqueous dispersions of UCNPs have shown the instability of luminescence over time due to particle dissolution-related effects. This can be especially detrimental for single-particle experiments. However, this effect has never been estimated at the individual particle level. Here, the luminescence response of individual UCNPs under aqueous conditions is investigated by quantitative wide-field microscopy. The particles exhibit a rapid luminescence loss, accompanied by large changes in spectral response, leading to a considerable heterogeneity in their luminescence and band intensity ratio. Moreover, the dissolution-caused intensity loss is not correlated with the initial particle intensity or band ratio, which makes it virtually unpredictable. These effects and the subsequent development of their heterogeneity can be largely slowed down by adding millimolar concentrations of sodium fluoride in buffer. As a consequence, the presented data indicate that microscopy experiments employing UCNPs in an aqueous environment should be performed under conditions that carefully prevent these effects.

Quantitative assessment of energy transfer in upconverting nanoparticles grafted with organic dyes.

Upconverting nanoparticles (UCNPs) are luminophores that have been investigated for a multitude of biological applications, notably low-background imaging, high-sensitivity assays, and cancer theranostics. In these applications, they are frequently used as a donor in resonance energy transfer (RET) pairs. However, because of the peculiarity and non-linearity of their luminescence mechanism, their behavior as a RET pair component has been difficult to predict quantitatively, preventing their optimization for subsequent applications. In this article, we assembled UCNP-organic dye RET systems and investigated their luminescence decays and spectra, with varying UCNP sizes and quantities of dyes grafted onto their surface. We observed an increase in RET efficiency with lower particle sizes and higher dye decoration. We also observed several unexpected effects, notably a quenching of UCNP luminescence bands that are not resonant with the absorption of organic dyes. We proposed a semi-empirical Monte Carlo model for predicting the behavior of UCNP-organic dye systems, and validated it by comparison with our experimental data. These findings will be useful for the development of more accurate UCNP-based assays, sensors, and imaging agents, as well as for optimization of UCNP-organic dye RET systems employed in cancer treatment and theranostics.