Nanoformulations offer multiple advantages over conventional drug delivery, enhancing solubility, biocompatibility, and bioavailability of drugs. Nanocarriers can be engineered with targeting ligands for reaching specific tissue or cells, thus reducing the side effects of payloads. Following systemic delivery, nanocarriers must deliver encapsulated drugs, usually through nanocarrier degradation. A premature degradation, or the loss of the nanocarrier coating, may prevent the drug’s delivery to the targeted tissue. Despite their importance, stability and degradation of nanocarriers in biological environments are largely not studied in the literature. Here we review techniques for tracing the fate of nanocarriers, focusing on nanocarrier degradation and drug release both intracellularly and in vivo. Intracellularly, we will discuss different fluorescence techniques: confocal laser scanning microscopy, fluorescence correlation spectroscopy, lifetime imaging, flow cytometry, etc. We also consider confocal Raman microscopy as a label-free technique to trace colocalization of nanocarriers and drugs. In vivo we will consider fluorescence and nuclear imaging for tracing nanocarriers. Positron emission tomography and single-photon emission computed tomography are used for a quantitative assessment of nanocarrier and payload biodistribution. Strategies for dual radiolabelling of the nanocarriers and the payload for tracing carrier degradation, as well as the efficacy of the payload delivery in vivo, are also discussed.

DNA modifications can be used to monitor pathological processes. We have previously shown that estimating the amount of the main DNA epigenetic mark, 5-methylcytosine (m⁵C), is an efficient and reliable way to diagnose brain tumors, hypertension, and other diseases. Abnormal increases of reactive oxygen species (ROS) are a driving factor for mutations that lead to changes in m⁵C levels and cancer evolution. 8-oxo-deoxyguanosine (8-oxo-dG) is a specific marker of ROS-driven DNA-damage, and its accumulation makes m⁵C a hotspot for mutations. It is unknown how m⁵C and 8-oxo-dG correlate with the malignancy of gliomas. We analyzed the total contents of m⁵C and 8-oxo-dG in DNA from tumor tissue and peripheral blood samples from brain glioma patients. We found an opposite relationship in the amounts of m⁵C and 8-oxo-dG, which correlated with glioma grade in the way that low level of m⁵C and high level of 8-oxo-dG indicated increased glioma malignancy grade. Our results could be directly applied to patient monitoring and treatment protocols for gliomas, as well as bolster previous findings, suggesting that spontaneously generated ROS react with m⁵C. Because of the similar mechanisms of m⁵C and guanosine oxidation, we concluded that 8-oxo-dG could also predict glioma malignancy grade and global DNA demethylation in cancer cells.

Understanding the interplay between nanoparticles (NPs) and cells is essential to designing more efficient nanomedicines. Previous research has shown the role of the cell cycle having impact on the efficiency of cellular uptake and accumulation of NPs. However, there is a limited investigation into the biological fate of NPs in cells that are permanently withdrawn from the cell cycle. Here we utilize senescent WI-38 fibroblasts, which do not divide and provide a definitive model for tracking the biological fate of silica nanoparticles (SiNPs) independent of cell cycle. We use several methods to measure the cellular uptake kinetics and intracellular retention of SiNPs, including confocal laser scanning microscopy (CLSM), flow cytometry, and transmission electron microscopy (TEM). We demonstrate that SiNPs readily enter into senescent cells. Once internalized, SiNPs do not exit and accumulate in the cytoplasm for long term. Our study provides a basis for future development of NP-based tools that can detect and target senescent cells for therapy.

Fluorogenic probes capable of distinguishing enzyme activity between species are crucial for precise detection in complex biological environments. Herein, we present 5-(Pentafluorobenzoylamino) Fluorescein di-β-D-galactopyranoside (PFB-FDG), a substrate selectively activated by human β-galactosidase (β-gal) without cross-reactivity with bacterial β-gal. We tested PFB-FDG under various control conditions using purified enzymes and live cells to demonstrate that it does not activate in the presence of bacterial β-gal. Moreover, molecular docking studies suggest that the selectivity results from steric hindrance within the smaller binding pocket of bacterial β-gal, in contrast to the larger binding pocket of the human enzyme. This species-selective probe represents a major advancement, with its unique properties providing a valuable tool for future studies on human-bacterial interactions.

Introduction: Fluorescent labeling is a widely used method for visualizing and tracking materials. 5-(Pentafluorobenzoylamino) fluorescein (PFB-F) is a dye in which the pentafluorobenzoylamino group substituent functions as a reactive linker. While PFB-F has been used for labeling biomolecules, its application in labeling surfaces has not been explored yet. Here, we report straightforward method for attaching PFB-F to amine-modified material surfaces requiring only dimethyl sulfoxide (DMSO) solvent and room temperature (RT). Methods: We conducted nuclear magnetic resonance (NMR) and thin layer chromatography (TLC) analysis on a mock reaction involving PFB-F and N-butylamine to evaluate the process of attachment via the PFB linker. PFB-F was also reacted with poly-L-lysine to evaluate conjugation to polymers bearing primary amine groups. Silica nanoparticles (SiNPs) labeled with PFB-F were administered to nematode worms, followed by confocal fluorescence microscopy. Results: NMR and TLC analysis confirmed covalent bonding between PFB-F and primary amines via nucleophilic aromatic substitution (S_NAr) involving displacement of at least one fluorine atom. PFB-F was successfully conjugated to poly-L-lysine, demonstrating efficient reaction with polymers containing primary amine groups. SiNPs labeled with PFB-F were ingested by nematode worms and successfully tracked in vivo. Discussion: These results demonstrate that PFB-F is a versatile and effective reagent for stable surface labeling. This study paves the way for broader applications of PFB-F in material design.

Glioblastoma remains one of the most aggressive primary brain tumors, characterized by rapid progression, therapeutic resistance, and poor prognosis. Dexamethasone is routinely administered to patients with glioblastoma to reduce peritumoral edema and improve neurological symptoms. However, its broader molecular effects on tumor-cell biology and treatment response remain incompletely understood. This study examined the effects of dexamethasone alone and in combination with temozolomide, valproic acid, or metformin on glioblastoma cell viability, real-time proliferation, senescence-associated β-galactosidase activity, cell-cycle distribution, global DNA methylation, and oxidative DNA damage. Dexamethasone induced concentration- and time-dependent changes in global DNA methylation in glioma cells. In T98G cells, shorter exposure increased 5-methylcytosine, whereas longer incubation or selected combined treatment conditions reduced methylation, suggesting dynamic epigenetic remodeling. Low dexamethasone concentrations were associated with increased 5-methylcytosine content, also in temozolomide-containing conditions. In contrast, higher dexamethasone concentrations reduced global methylation. That effect was cell-line dependent. In SW1783 cells, high-dose dexamethasone caused a reduction in 5-methylcytosine amount as well as an increase in 8-oxo-2′-deoxyguanosine level. Dexamethasone also reduced real-time T98G cell proliferation after prolonged exposure, while temozolomide was the main compound associated with cell-cycle changes in T98G cells. Metformin displayed comparatively limited cytotoxicity under the tested conditions. Beyond dexamethasone’s well-established anti-edematous and anti-inflammatory role, it may also modify glioma redox and epigenetic states and influence cellular responses to temozolomide, valproic acid, and metformin. When considering these findings it is necessary to take into account a number of limitations. The upper limits of the DEX, TMZ, and MF concentration ranges were one to three orders of magnitude higher than the brain or CSF exposure levels that have been reported, and the experiments were carried out at standard culture oxygen concentrations (about 18–21% O₂), which are much higher than the oxygen levels found in brain tissue (about 2–6% O₂). Furthermore, the conclusions drawn from the T98G experiments only apply to this cell line and cannot be applied to glioblastoma without first being verified in other models.