With ever-increasing applications of nanoscale materials in the biomedical field, the impact of nanoparticle size on cellular uptake efficiency, dynamics, and mechanism has attracted numerous interests but still leaves many open questions. A combined “multiphoton imaging-UV/Vis spectroscopic analysis” method was applied for the first time for quantitative visualization and evaluation of the cellular uptake process of different-sized (15-, 30-, 50-, and 80-nm) gold nanoparticles (AuNPs). Quantitative analysis of the size effect on cellular uptake behavior of AuNPs from a stack of three-dimensional multiphoton laser scanning microscopy images is obtained. The technique allows for differentiating AuNPs present in external and internal subcellular components, giving detailed information for elucidating cellular uptake dynamics without particle labeling. The data show that the internalization extent of AuNPs is highly dependent on particles’ sizes and incubation time. Due to sedimentation, 50- and 80-nm AuNPs are taken up to a greater extent than 15- and 30-nm particles after exposure for 24 h. However, the smaller particles’ uptake velocity is significantly faster in the first 10 h, indicating a disparity in uptake kinetics for different-sized AuNPs. The finding from this study will improve our understanding of the cellular uptake mechanisms of different-sized nanoparticles and has great implications in developing AuNP-based drug carriers with various sizes for different purposes.
Respiratory mucus is one of the main barriers for nanoparticle-based pulmonary delivery systems. This holds true especially for lung diseases like cystic fibrosis, where a very tenacious thick mucus layer hinders particle diffusion to the lung epithelium or the target area. Typically, mean square displacement of particles is used for mobility evaluation. In contrast, our objective is to develop a feasible technique to track directed particle penetration as a prerequisite for efficient pulmonary nanotherapy. Therefore, particle diffusion in artificial mucus was monitored based on confocal laser scanning microscopy (CLSM) and particle-mucus interaction was observed. As pharmaceutical relevant and benign materials, solid lipid nanoparticles (SLNs) were prepared by hot-melt emulsification using glyceryl behenate and different stabilizing agents such as poloxamer-407, tween-80, and polyvinyl alcohol (PVA). The diffusion of labeled SLNs in stained artificial sputum representing CF-patient sputum was verified by 3D time laps imaging. Thus, the effect of coating, particle size and mucus viscosity on nanoparticle diffusion was studied. Using image analysis software "Image J", the total fluorescent signal after 30 min in case of poloxamer-coated SLNs was 5 and 100 folds higher than tween- and PVA-coated SLNs, respectively. Nevertheless, increasing mucus viscosity reduced the diffusion of tweencoated SLNs by a factor of 10. Studying particle-mucus interaction by CLSM can be considered a promising and versatile technique.