We have used a silica – PEG based bionanoconjugate synthetic scheme to study the subtle connection between cell receptor specific recognition and architecture of surface functionalization chemistry. Extensive physicochemical characterization of the grafted architecture is capable of capturing significant levels of detail of both the linker and grafted organization, allowing for improved reproducibility and ultimately insight into biological functionality. Our data suggest that scaffold details, propagating PEG layer architecture effects, determine not only the rate of uptake of conjugated nanoparticles into cells but also, more significantly, the specificity of pathways via which uptake occurs.

Inter-laboratory comparison of nanoparticle size measurements using dynamic light scattering and differential centrifugal sedimentation. NanoImpact, (2018).

Nanoparticles (NPs) functionalized with two active targeting ligands have been proposed in drug delivery for their promising capability to stimulate different pathways with one object. Due to the multivalency, the construction and analysis of the effective surface of such bifunctional nanoparticles, however, is significantly more complex than for nanoparticles bearing only one ligand. Here, we optimize construction and analysis of bifunctional NPs containing recognizable combinations of human serum albumin (HSA), transferrin (Tf), and epidermal growth factor (EGF) on fluorescent silica NPs grafted via common polyethylene glycol (PEG) linkers as a model system. Combined with an overall protein quantification, a mapping of exposed recognizable sequences using monoclonal antibodies conjugated to gold nanoparticles (AuNPs) or quantum dots (QDs) for enhanced spectroscopic and microscopic detection revealed that active protein sequences can be one to two orders of magnitude lower than overall conjugated proteins while possessing specific cellular recognition. In addition, we found that common conjugation strategies lead to a large excess of non-specifically compared to covalently bound ligands and instabilities that may impact targeting. These can be avoided by certain synthetic conditions presented here for effective exploitation of multivalent surfaces in nanomedicine.

Observing structural integrity of nanoparticles is essential in bionanotechnology but not always straightforward to measure in situ and in real-time. Fluorescent labels used for tracking intrinsically nonfluorescent nanomaterials generally do not allow simultaneous observation of integrity. Consequently, structural changes like degradation and disassembly cannot easily be followed in situ using fluorescence signals. We show that thioflavin T (ThT), a fluorophore and molecular rotor known to tag specific fibril structures in amyloids, can "label" the structural integrity of widely used and intrinsically nonfluorescent, silica nanoparticles (SiNPs). Entrapment of ThT in SiNPs controls the fluorohphore's relaxation pathway and leads to a red-shifted fluorescence spectrum providing real time information on SiNP integrity. The dynamic change of ThT fluorescence during degradation of doped SiNPs is found much higher than that of common labels fluorescein and rhodamine. Degradation kinetics of core-shell structures recorded by ThT fluorescence and light scattering prove the capability to clearly distinguish structural features during SiNPs degradation and allow obtaining degradation kinetics in vitro, in biological media, in serum, and in cells. The effect is transferable to different types of materials, here shown for ThT incorporated SiNPs with tightly tailorable sizes (9-100 nm), poly(lactic-co-glycolic acid) (PLGA) nanoparticles, poly(9-vinylcarbazole) (PVK) nanoparticles, and iron-doped-SiNPs (FeSiNPs). We thus suggest molecular rotors such as ThT as additional labels to effectively and easily sense nanoparticle structural status in situ and to enhance understanding and development of programmed nanoparticle disassembly in bionanotechnology.

The characterization of nanoparticles in dispersions, in particular measuring their size and size distribution, is a prerequisite before they can be used in toxicological testing. Such characterization requires reliable methods with good reproducibility. The aim of this study was to evaluate the reproducibility, and thus the potential of Dynamic Light Scattering (DLS) for nanoparticle size determination. DLS is easy to use and well established in most nanotoxicology laboratories. However, reproducibility and in particular variability between measurements done using different instrumental setups have not been addressed systematically before. Here we performed initial experiments with rather monodisperse dispersions of spherical particles in water. Significant discrepancies in the measured distributions were obtained with different DLS instruments, especially when fitting the data using mathematical inversion methods. Significant errors can be made due to different settings being used for fitting the data. These were even more prominent when working with dilute dispersions of very small particles. Our study has identified several important points to be taken into consideration in order to overcome possible issues in measurement and analysis of nanoparticles using DLS.In practice, however, nanoparticles may have significant polydispersities and/or can be non-spherical. We extend the comparative work on spherical particles, to show how to characterize polydisperse and/or high aspect ratio particles using DLS instruments.