Upconversion nanoparticles-based photodynamic nanotheranostic agents (UCNPs-PDT) have received great interest due to improved tissue penetration, weak autofluorescence, and low biotoxicity. However, conventional UCNPs-PDT are often limited by low energy transfer efficiency from UCNPs to photosensitizer (PS) molecules and insufficient generation and limited diffusion distance of reactive oxygen species (ROSs). Herein, an “all in one” nanotheranostic agent has been developed which has multicolor sandwich-structured UCNPs (SWUCNPs) as the core, a thin silica layer with a mitochondria-targeted group for loading dual PS as the medium layer, and polyethylene glycol–folic acid (PEG-FA) chains as the outer layer. Multicolor SWUCNPs simultaneously achieve two-photon fluorescence imaging and serve as energy donor for dual PS molecules. The thin luminescence layer and silica layer control most UCNPs activators and PS molecules in the effective energy transfer distance to guarantee a high energy transfer efficiency. Via FA-mediated endocytosis, the nanotheranostic agent is selectively endocytosed by cancer cells, is released from the endosome/lysosome, targets the mitochondria, and in situ produces ROSs under excitation from NIR, leading to significant mitochondria-mediated cell apoptosis. Furthermore, the established nanotheranostic agent shows tumor targetability, increased generation of ROSs, high PDT efficacy, significant cell apoptosis, minimal systemic cytotoxicity, and efficacious in vivo tumor inhibition.

Monitoring the fluctuation of hydroxyl radical (·OH) in the body can serve as an effective tool for the prediction of relative diseases; however, it is highly challenging due to the radical's short lifetime, high reactivity, and extremely low concentration. Sandwich structured lanthanide-doped upconversion nanoparticles (UCNPs) exhibit unique luminescence properties and great prospects in bioimaging. Nonetheless, their rather low luminescence efficiency and intensity are serious limitations for their application. Herein, we report on dual-activator codoped UCNPs with a core-multishell structure that greatly improve the luminescence intensity and lifetime by 46-fold and 2.6-fold, respectively, compared to those of the monoactivator doped sandwich structured UCNPs. Moreover, emitting ions in the designed core-multishell (CMS)-UCNPs were confined in a homogeneous and thin shell layer (∼2 nm); thus, the luminescence resonance energy transfer (LRET)-based CMS-UCNPs@azo dye nanoprobe exhibited a largely shortened energy transfer distance and a pronounced luminescence quenching yield (97%), affording the nearly zero background signal and achieving an ultrahigh sensitivity for the detection of ·OH (with limit of quantitation (LOQ) of 0.10 fM). With good biocompatibility, low biotoxicity, and enhanced luminescence intensity and lifetime, the developed nanoprobe was competent in monitoring the subtle fluctuation of ·OH concentration both in vitro and in vivo.

Intracellular [Ca ] and pH have a close relationship, and their abnormal levels can result in cell dysfunction and accompanying diseases. Thus, simultaneous determination of [Ca ] and pH can more accurately investigate complex biological processes in an integrated platform. Herein, multicolor upconversion nanoparticles (UCNPs) were prepared with the advantages of no spectral overlapping, single NIR excitation wavelengths, and greater tissue penetration depth. The upconversion nanoprobes were easily prepared by the attachment of two fluorescent dyes, Fluo-4 and SNARF-4F. Based on the dual luminescence resonance energy transfer (LRET) process, the blue and green fluorescence of the UCNPs were specially quenched and selectively recovered after the detachment and/or absorbance change of the attached fluorescent dyes, enabling dual detection. Importantly, the developed nanoprobe could successfully be applied for the detection of [Ca ] and pH change in adenosine triphosphate (ATP) and ethylene glycol tetraacetic acid (EGTA) stimulation in living cells.