Interaction of 4 allotropic modifications of carbon nanoparticles with living tissues

Environmental pollution and technological progress lead to carbon nanoparticles that pose a serious health risk. They are present in soot, dust, and printing toner and can also be formed during grinding and cutting. Human neutrophils are able to sequester foreign material by formation of neutrophil extracellular traps (NETs), a process that can cause a strong inflammatory response. In the current work we compared proinflammatory properties of different carbon-based nanostructures: nanodiamonds, graphene oxide, fullere­nes C60 and carbon dots. We tested adjuvant properties of carbon nanoparticles in a murine immunization model by investigating humoral (specific IgG and IgM antibodies) and cellular (delayed type hypersensitivity) immune responses. The ability of NETs to sequester nanoparticles was analyzed in a mouse air pouch model and neutrophil activation was verified by in vivo tracking of near-infrared labeled nanodiamonds and ex vivo fluorescent assays using human blood-derived neutrophils. All carbon nanoparticles exhibited proinflammatory adjuvant-like properties by stimulating production of specific IgG but not IgM antibodies (humoral immune response). The adjuvant-like response decreased in this order: from nanodiamonds, graphene oxide, fullerenes C60 to carbon dots. None of the studied carbon nanoparticles triggered a delayed type hypersensitivity reaction (cellular immune response). Nanodiamonds and fullerenes C60 were sequestrated in the body by NETs, as confirmed in the air pouch model and by in vivo fluorescent tracking of near-infrared labeled nanodiamonds

Simple two-step covalent protein conjugation to PEG-coated nanocrystals

Covering of nanocrystals (NC) with a polyethylene glycol (PEG) envelop is a common way to increase their hydrophilicity, and compatibility with bio-systems, including increased retention time in the body. Colloidal semiconductor NC, also known as quantum dots (QD), particularly benefit from covering with PEG due to passivation of the inorganic core, while maintaining physical properties of the core. Despite many advantages of covering the surface with PEG, the covalent attachment of protein to hydroxyls of PEG is complicated. Here we propose a simple two-step approach for modification of PEG residues with subsequent covalent attachment of proteins. We were able to achieve specific NC targeting by means of attached protein as well as preserve their optical parameters (fluorescence intensity) in chemical reaction conditions. In the optimized protocol, ensuring removal of chemical byproducts by dialysis, we were able to omit the need for centrifugation (usually a limiting step due to particle size). The obtained NC-protein conjugate solutions contained 0.25x of initial unmodified NC amount, ensuring a low dilution of the sample. During all reactions the pH range was optimized to be between 6 to 8. The proposed approach can be easily modified for covalent targeting of different PEG-covered nanocomposites with proteins