Polysaccharide coated upconversion nanoparticles for uptake and imaging in cells
Fluorescent nanomaterials are promising for combining diagnostics and therapeutics into a single system, leading to more precise and capable tools for early diagnosis and treatment of diseases. Upconversion nanoparticles (UCNPs) are an exceptionally photostable nanomaterial that is ideal for bio-imaging as they are excited by deep-penetrating near-infrared light but emit in ultraviolet, visible or near-infrared wavelengths. We have created novel polysaccharide coated UCNPs via ligand exchange that are highly hydrophilic and colloidally stable due to the presence of a coating of negatively charged sugars. The surface functionalisation of 4% Tm doped NaYF4 UCNPs was achieved using the ligand exchange method where UCNPs were mixed with Fucoidan, Colominic acid (Col.A) or Chondroitin sulphate (Cho.S) in a two -phase solution with overnight shaking. Zetasizer and dynamic light scattering (DLS) measurements were used to validate the surface functionalisation along with lectin binding assays. Zeta potential indicated the change in charge from neutral to highly negatively charge whereas DLS measurements indicated a change in the size of particles ranging from 70nm – 110nm for various polysaccharide UCNPs. Aleuria aurantia lectin - Fluorescein (AAL-FITC) bound to Fucoidan-UCNPs and co-localised luminescence indicated the presence of these sugars on the UCNPs. Transmission electron microscopy imaged UCNPs before and after functionalisation.
Cellular uptake studies were performed using different human cell lines such as SH-SY5Y (Epithelial Neuroblastoma), U-87 MG (Epithelial Glioblastoma) and hCMEC/D3 (Brain Endothelial) cell lines to visualise uptake of the different polysaccharide coated UCNPs using confocal microscopy. The polysaccharide nanoparticles were internalised differently in the cell lines with and without LPS induced inflammation, indicating the possible involvement of specific sugar receptors in the uptake mechanism. Colominic acid coated UCNPs were imaged at single particle resolution in PC12 (pre-neuronal cells) using the super resolution techniques of Super-linear excitation-emission microscopy (SEE) and Stimulated emission depletion (STED). Analysis of Fucoidan-UCNP nanoparticle uptake showed increasing UCNP uptake into macrophage cells over 16 hrs. This uptake was inhibited by pre-incubation with the monosaccharide component of fucoidan, indicating a specific fucose receptor mediated mechanism for Fucoidan-UCNP uptake. A biodistribution study in vivo indicated that Fucoidan-UCNPs are safe, non-toxic and allow for further targeted therapy and drug delivery applications.
Overall, we have demonstrated that Fucoidan, Col.A and Cho.S polysaccharides convert UCNPs into a hydrophilic state while maintaining their stability and dispersion in aqueous phase. These sugar-coated UCNPs were shown to have differential uptake into various cells, and that this uptake changed with inflammation, and enabled single particle super resolution microscopy inside cells. Thus, these biocompatible polysaccharide-coated luminescent UCNPs have the potential to explore intracellular mechanisms via live cell quantitative imaging and targeted delivery.